Rozprawy doktorskie na temat „Regenerative engineering”

Regenerative medicine is an emerging field that aims to treat injury and disease by harnessing and augmenting the body’s innate capacity for tissue regeneration. Many of the strategies developed in this field have relied extensively on the principles of tissue engineering, a set of methods that bring together cells, cellular signals and material scaffolds to repair or replace biological tissue. While the number of novel tissue engineering strategies continues to rapidly expand, the innovations underlying these solutions often fail to consider the key technical, manufacturing, and regulatory barriers that prohibit these technologies from suitable use in humans. As a result, the field of tissue engineering has one of the lowest rates of clinical translation amongst medical research. To address this, this thesis examines each of the prominent components of the tissue engineering practice and develops tools and strategies that enable the development of solutions with high translational potential. The collective findings of these works propose tools and solutions applicable within the major facets of tissue engineering that may help to lay the groundwork for future therapies with high clinical probability in a number of regenerative medicine applications.

Tissue engineering with fibrous scaffolds is emerging as a major research area in the field of regenerative medicine. The major themes pursued in this thesis are the study of the cellular response to nanofibrous constructs, the role of nanofibres in the engineering of synthetic scaffolds, and the development of technology to facilitate the fabrication of nanofibrous scaffolds with controlled architectures. Cells cultured on multi‐walled carbon nanotubes displayed reduced proliferation and altered cytoskeletal development, thought to be due to the undermining of the maturation of focal adhesions. Development of an electrospinning chamber enabled the creation of poly(methyl methacrylate), poly(lactic acid) and poly(caprolactone) fibres for the study of cellular response to nano‐ and macro‐fibrous scaffolds. Cell attachment and organisation on the electrospun fibres was visualised using scanning electron microscopy, oblique microscopy and live cell microscopy. It was found that the incorporation of nanofibres into scaffolds restricts the maturation of focal adhesions which modulates cytoskeletal formation. This can be used to restrict the migration and the proliferation of attachment dependant cells such as osteoblasts or maintain the differentiation of cells such as chodrocytes. To scale up electrospun fibre production, use of rotating collectors, multi‐jet spinning and secondary electrodes to focus the spinning were investigated. Further to this, development of an array of focusing electrodes to control, stabilise and deflect the jet was also investigated towards the creation of a rapid‐prototype electrospinning system. The secondary electrode array was found to reduce the spreading of the jet to a spot diameter of 10mm and charged deflection plates successfully redirecting the position of the jet as it arrived at the collector.

Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting. Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process. In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect. In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture. Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture. In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.

Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting. Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process. In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect. In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture. Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture. In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.

Existing, dermal, regenerative scaffolds facilitate dermal repair and wound healing of severe burn injuries; however, new tissue is often functionally, mechanically and aesthetically abnormal due to irregular deposition of new extracellular matrix. In the present study two novel, elastin-containing scaffolds were developed, characterised and examined both in vitro and in vivo aiming to minimise wound contraction, improve scar appearance and increase skin elasticity post-healing. The first types of scaffolds were electrospun from a triple polymer solution of collagen, elastin and poly(ϵ-caprolactone) (CEP). Two scaffolds were chosen for characterisation: CEP 1 was fabricated using a 1.5 % (w/v) collagen, 12 % (w/v) elastin and 1.5 % (w/v) poly(ϵ-caprolactone) (PCL) solution, a flow rate of 3 mL/h, an air gap of 15 cm and an applied electric potential of 25 kV; and CEP 2 was electrospun using a 2 % (w/v) collagen, 12 % (w/v) elastin and 1 % (w/v) PCL solution at 1 mL/h, 20 cm and 20 kV. In vitro cell studies using human, dermal fibroblasts (HDFs) and immortalised, human keratinocytes (HaCaTs) revealed CEP 1 and CEP 2 supported cell-seeding and cell proliferation with significantly higher proliferation of both cell types on CEP 1. Additionally, subcutaneous implant studies in mice revealed minimal inflammation in response to both scaffolds with CEP 1 vascularised by week 2 post-surgery. However, CEP 1 was rapidly biodegraded after 2 weeks. Collagen deposition was observed in encapsulating tissue and new tissue with consistent collagen expression over 24 weeks. The second type of scaffold investigated was an elastin-modified version of the commercial, dermal substitute Integra Dermal Regeneration Template (IDRT). Elastin-IDRT (EDRT) was developed by inclusion of 10% human tropoelastin and then investigated in comparison with IDRT. Morphological analysis by scanning electron microscope and mechanical characterisation revealed EDRT had significantly enlarged pores, higher porosity and increased deformability. Higher cell seeding efficiency of HaCaTs on EDRT was observed compared to IDRT but cell proliferation rate was found to be similar over 28 days. HDFs displayed increased cell growth rate on EDRT over 28 days compared to IDRT. Enhanced and accelerated HDF infiltration of EDRT was also visualised with complete infiltration by day 14 post-seeding. An in vivo, mouse, subcutaneous implant model showed that EDRT induced minimal inflammation. Gene expression of mouse collagen was consistent over 24 weeks with non-significant increases in elastin expression from weeks 2 and 4. One-step grafting demonstrated similar contraction between EDRT-, IDRT- and autografted wounds with final contraction around 40 % compared to 100 % in open wounds. EDRT displayed significantly accelerated, early-stage angiogenesis with higher vascularisation than IDRT-grafted, autografted or open wounds 2 weeks post grafting. By week 4 EDRT- and IDRT-grafted wounds had similar levels of vascularisation which were higher than autografted and open wounds. EDRT showed improved mechanical performance, supported enhanced cell interactions in vitro and accelerated angiogenesis in vivo. In summary, investigated scaffolds demonstrated properties that could potentially improve burn wound healing. The inclusion of elastin in scaffolds produced by either electrospinning or lyophilisation improved HDF infiltration and supported formation of a confluent layer of HaCaTs which could result in increased pliability of new skin and accelerated wound healing. In EDRT elastin improved scaffold porosity, pore size and accelerated angiogenesis in vivo indicating EDRT can facilitate and improve wound remodelling. Further investigation of both scaffolds is warranted especially due to the vascular inductive effects of EDRT and the synchronous spatial and temporal biodegradation of CEP 2 observed in vivo.

Degenerative conditions of the spine are a major public health problem, leading to severe back pain, reduced quality of life and chronic disablement in a proportion of sufferers. For some of these patients, spinal fusion surgery is a treatment that can alleviate back pain and restore normal function. However, limitations in the availability of graft material mean that alternative grafts are needed and tissue-engineering approaches have been employed. Using a novel self-organising collagen scaffold combined with nano-hydroxyapatite and chondroitin sulphate and by employing the latest materials techniques, I have studied the osteogenic capability of a biomimetic graft for use in spinal fusion surgery. The mineralised collagen scaffold has compressive strength comparable to human cancellous bone and can support the proliferation of viable human mesenchymal stem cells. This porous scaffold can be combined with human mesenchymal stem cells to further promote bone growth, as evidenced by an upregulation in the levels of bone-forming genes and mineralisation of the scaffold. This scaffold can act as a carrier system for BMP-2, with wider application for other growth factors or drugs, providing sustained release when fabricated as a layer-by-layer scaffold. An alternative bone substitute for use in spinal surgery has been designed and characterised, with exciting potential for use in vivo.

The aim of this study was to investigate the stability of positional identity markers and phenotypic differences in isolated osteoblasts from distinct anatomic regions. In addition, the ability of heterotypic co-cultures to reprogramme site-specific Hoxa gene expression also tested. Rat osteoblastic cells from femurs and calvariae were harvested as matched pairs of cultures from 4 male rats. Cells were expanded extensively in medium supplemented with FGF-2, and were shown to maintain their osteoblastic phenotype as characterised by alkaline phosphatase (ALP) staining, osteopontin (OPN), osteocalcin (OCN) expression and osteoblast-associated gene expression in long term culture. Gene expression of cells was determined by quantitative RT-PCR. Differences in Hoxa gene expression as markers of positional identity were maintained for up to at least 10 passages, with calvarial cells remaining Hoxa-ve throughout. The transcription factors Msx2 and Irx5 were consistently more highly expressed in calvarial cells, whereas Tbx3 expression was elevated in femoral cells. Expression of the osteoblast-associated genes Bglap and Sppl were elevated in femoral cells, and also associated with increased osteopontin secretion and bone nodule formation. Runx2 was elevated in calvarial cells. Cells were also pre-labelled with fluorescent vital staining and co-cultured for 7 days prior to separating by fluorescence activated cell sorter to investigate the possibility of re-programming of Hoxa negative cells by direct contact with Hoxa +ve cells. However no evidence was seen of modulation of positional identity genes and phenotypes in these heterotypic cultures. In conclusion, the results demonstrate persistence of expression of positional identity gene markers and phenotypic differences between femoral and calvarial osteoblasts for prolonged periods in culture. These data suggest that these differences in regionally defined osteoblasts are inherently programmed in the cells as a result of their embryological position. The results may have considerable implications when considering the transplantation of autologous cells in tissue engineering.

The Super- Regenerative Reception concept was developed in the early 1900s. The evolution of Heterodyne Reception has forced it into oblivion. However, recent research and development has emphasized its use in short distance and low power wireless applications. The results indicate extremely low power and high gain with small circuit area.

The study herein focuses on a receiver using a Super Regenerative Oscillator to decrypt 8-PSK Signals. Demodulation without the use of a Local Oscillator, a Phase Locked Loop or an Analog to Digital Converter is proposed. The architecture of encoding is presented which is in turn used to generate the necessary input at the receiver. The receiver involves a combined Low Noise Amplifier Super- Regenerative Oscillator architecture in a single stack configuration for the feature of current reuse. Simulation results are presented which confirm the theory. Finally, a comparative study is presented for various modulation techniques including Quadrature Phase Shift Keying and 8-Phase Shift Keying.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.
Includes bibliographical references (leaves 28-29).
This work develops a method for capturing some of the kinetic energy ordinarily lost during braking on bicycles to power LED safety flashers. The system is designed to eliminate: (a) battery changing in popular LED flashers, and (b) the "generator drag" associated with battery-less human-powered bicycle lights and flashers. System sizing, mechanical design considerations, potential end-user factors, and a model for braking frequencies in urban settings are discussed. With the urban commuter cyclist in mind as a potential user of the regenerative braking system, custom direct-pull brake calipers (or "V-Brakes") were designed and manufactured to include both conventional friction pads in addition to a DC motor to be used as a generator for kinetic energy capture. The energy captured by the DC motor during braking is passed through a full wave bridge to a bank of Nickel-Cadmium batteries at an efficiency of 79%. The output of the full wave bridge and the batteries are connected in parallel with a step-down switching voltage regulator, which insulates the LED safety flasher from voltage spikes due to braking at high cycling speeds. The performance of the final prototype was evaluated at cycling speeds ranging from 8 to 19 mph and braking frequencies ranging from 2 to 8 operations/stops per mile of travel.
(cont.) From the mean power flow (charging) into the batteries per unit distance of travel and the power required by LED safety flashers, the effectiveness of the system at each speed and stopping frequency is examined. For cyclists traveling at average speeds of 10 mph or higher, the LED safety flashers can be powered continuously for stopping frequencies of 8 times per mile and semi-continuously (> 50% of the time) for stopping frequencies of at least 4 times per mile. As such, the system is determined to be potentially useful to urban commuter cyclists, who frequently perform braking operations at regularly spaced intersections and traffic signals, and who regularly travel by bicycle in low-light conditions (dawn or dusk), though usually less than 50% of the time.
by Ian M. Collier.
S.B.

Fracture non-unions and bone defects represent a recalcitrant problem in the field of orthopaedic surgery. Although the current gold-standard treatment, autologous bone grafting, has a relatively high success rate, the technique is not without serious problems. The emerging field of regenerative medicine may have the potential to provide an alternative treatment. One promising strategy involves the delivery of both cells and multiple growth factors with different release profiles. A range of scaffolds was developed from Poly( -caprolactone) (PCL), Poly(lactideco- glycolide) (PLGA), and two blends of PCL (Mn 42,500) and PLGA. The scaffolds were manufactured utilising a novel modified fused deposition modelling system, using polymer/dichloromethane solutions. The scaffolds were found to have pore sizes suitable for bone regenerative medical applications (373±9.5 μm in the Ydirection and 460±13 μm in the X-direction). However, the scaffolds were found to be only 52±3 μm in height. This means that the two-layer scaffolds were relatively flat. This was undesirable, as direct control of the complete 3D geometry was the favoured strategy, though it may not be a necessary requirement. Five scaffold coatings were also developed from alginate, chitosan (crosslinked using sodium hydroxide or tripolyphosphate), Type-I collagen and Type-A gelatin. The scaffold coatings were screened in vitro for their cell-compatibility with human marrow stromal cells (hMSCs), human osteoblasts and MG63 cells. This was assessed using an assay for cell death, and assessing total cell counts. From these studies, Type-I collagen was found to be the optimum coating. For hMSCs, their death rates were found to be 19.1±6.3% for alginate, 5.3±3.6% and 2.9±1.4% for chitosan crosslinked with tripolyphosphate and sodium hydroxide respectively, compared to 0.11±0.07% for Type-I collagen, and 0.15±0.13% and 0.16±0.12% for 0.1% and 0.2% gelatin respectively. Type-I collagen was found to be the most cellcompatible coating, as it was consistently associated with higher cell counts than Type-A gelatin. Similarly, PCL scaffolds vacuum dried for 1 hr were found to be cell-compatible. No detectable clinically significant difference was found in either total cell counts, or the proportion of cell death in; hMSCs exposed to PCL scaffolds processed with dichloromethane, hMSCs either exposed to scaffolds known to be biocompatible, or hMSCs cultured in the absence of scaffolds. When cell morphology was compared, scaffolds vacuum dried for 1 hr or more were found to have a similar morphology to the cells cultured in the absence of scaffolds. It was therefore concluded that a vacuum drying time of 1 hr was sufficient for cell-compatibility. The scaffold materials were screened both for their encapsulation efficiencies and release characteristics using the model drug, methylene blue. The encapsulation efficiency was found to be both relatively high and consistent for both Mn 42,500 and 80,000 PCL as well as PCL:PLGA 66:33, at 71±6%, 71±5%, and 78±10% respectively, relative to the low efficiencies recorded for both PCL:PLGA 66:33 and PLGA: 57±5% and 38±10% respectively. The release rate of methylene blue from PCL (Mn 42,500), was found to be relatively slow, controlled, and consistent between batches (between 21±2% and 20±3% released in the first 24 hr). Despite the release rate being consistent for PCL (Mn 80,000), the release rate was thought to be too high, since between 29±3% and 39±5% of the test compound was released in the first 24 hr period. The release rate of methylene blue from the PCL/PLGA blends (between 17±2% – 30±7% and 18±4% – 31±6% in the first 24 hr) and PLGA (between 7.1±3.4% – 9.3±2.9% in the first 24 hr) were found to be inconsistent, and low in the case of PLGA, even taking the different loading efficiencies into account. Therefore, PCL (Mn 42,500) was selected as the favoured candidate scaffold material. The loading content and release profiles from methylene blue loaded collagen scaffold coatings were also evaluated. The drug loading capacity was found to be suitable for use as a drug delivery system (65±5 μg/g of methylene blue per unit scaffold mass). The release of methylene blue was observed to be rapid (between 54±10% – 70±17% in the first 24 hr), which was thought to be desirable for the coating delivery system. Recombinant human bone morphogenetic protein-7 (rhBMP-7) was used as a representative growth factor of interest for bone regenerative medical applications. It was loaded in collagen scaffold coatings (CoatBMP 1.25) and encapsulated within PCL (Mn 42,500) scaffolds (ScaffBMP 1.25). Control coatings and scaffolds were designated CoatPBS and ScaffPBS respectively. Both delivery systems were found to release detectable quantities of rhBMP-7 (releasing 2.8±0.2 μg/g and 87±7 ng/g respectively in the first 24 hr), even after 14 days. The release rate of the growth factor from the scaffold coating was higher than that from the encapsulating scaffolds. However, the cumulative release profiles were found to deviate from the desired ideal release profiles, and burst release was observed from both delivery systems. Although differences were observed for the two delivery systems, this difference may not be of clinical significance. Nevertheless, scaffolds with less than ideal delivery properties may still be of potential clinical use. The bioactivity of the rhBMP-7 released from the test scaffolds was therefore assessed by quantifying the area of normalised ALP staining of hMSCs. The release of rhBMP-7 from the collagen coating of the PCL (Mn 42,500) scaffolds (CoatBMP 1.25ScaffPBS) was capable of statistically significantly increasing hMSC normalised ALP expression, although the actual differences were often relatively small. Therefore, at least a proportion of the growth factor released is likely to have been bioactive. The release from scaffolds encapsulating rhBMP-7 (CoatPBSScaffBMP 1.25) did not have this effect on the hMSCs, indicating that either the concentration released was too low, or the growth factor released was no longer bioactive. However, when the cells were seeded directly onto the scaffolds, the activity of ALP, normalised by a DNA assay, was statistically significantly increased for the CoatPBSScaffBMP 1.25 scaffolds, in hMSCs from all three test patient donors (by 35±10% on the control). ALP activity was also significantly increased in hMSCs from two of the three patients seeded onto CoatBMP 1.25ScaffBMP 1.25 scaffolds (by 39±10% on the control). ALP activity was only statistically significantly increased for one of the hMSC patients when seeded onto CoatBMP 1.25ScaffPBS scaffolds (by 35±14% on the control). The functional osteoinductive capacity of Type-I collagen coated PCL (Mn 42,500) scaffolds loaded with rhBMP-7 was assessed using C2C12 cells seeded onto the scaffolds, and quantified using qRT-PCR. The genes of interest were; Type-I collagen (Col1), osteopontin (OP), ALP, osteocalcin (OC) and runt related transcription factor 2 (Runx2). The CoatBMP 1.25ScaffPBS scaffolds had an early osteoinductive effect on the C2C12 cells, as ALP, OC and Runx2 were elevated during the first 2 days only, compared to the control (e.g. by 44±12%, 128±42%, 60±25% and 46±25% respectively at the 24 hr mark). The CoatPBSScaffBMP 1.25 scaffolds also had an osteoinductive effect on the cells, which was more sustained than that observed for the CoatBMP 1.25ScaffPBS group. While OP, ALP and Runx2 were up-regulated in the first 24 hr compared to the control (by 38±10%, 208±82% and 72±31% respectively), statistically significant up-regulation of the late marker OC was delayed until the 48 hr mark (by 73±49%). The effect was found to be sustained until day 7, when OC and Runx2 were both statistically significantly up-regulated compared to the control (by 151±91% and 93±27% respectively). The CoatBMP 1.25ScaffBMP 1.25 scaffolds were found to combine the early effect of the CoatBMP 1.25ScaffPBS scaffolds, with the more sustained effect of the CoatPBSScaffBMP 1.25 scaffolds. ALP, OC and Runx2 were all up-regulated at the 24 hr mark (by 312±56%, 329±39% and 96±25% respectively). This osteoinductive effect was sustained until day 7 when Col1, ALP and Runx2 were still up-regulated compared to the control (by 174±78%, 72±24% and 178±78% respectively). These data suggest that the scaffolds containing rhBMP-7 have a weak osteoinductive effect on the cells seeded onto them. The different delivery systems were found to affect the cells differently. The clinical significance of this was not assessed in these studies. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was used as a model drug to assess the feasibility of releasing lipid-soluble active factors from the scaffolds. This was assessed by quantifying the area of normalised ALP staining of hMSCs. The release of 1,25(OH)2D3 from the loaded collagen scaffold coatings and the encapsulating scaffolds significantly increased ALP expression compared to the control scaffold groups (by 115±28% and 69±25% respectively). Furthermore, ALP expression was significantly increased when the two delivery systems were used together, when compared to either delivery system on its own. These data suggest that the delivery of lipid-soluble active factors is feasible from collagen coated PCL scaffolds, and that the coating and encapsulating delivery systems are mutually compatible.

In recent years the optical domain has been traditionally reserved for node-to-node transmission with the processing and switching achieved entirely in the electrical domain. However, with the constantly increasing demand for bandwidth and the resultant increase in transmission speeds, there is a very real fear that current electronic technology as used for processing will not be able to cope with future demands. Fuelled by this requirement for faster processing speeds, considerable research is currently being carried out into the potential of All-optical processing. One of the fundamental obstacles in realising All-optical processing is the requirement for All-optical buffering. Without all-optical buffers it is extremely difficult to resolve situations such as contention and congestion. Many devices have been proposed to solve this problem however none of them provide the perfect solution. The subject of this research is to experimentally demonstrate a novel all-optical memory device. Unlike many previously demonstrated optical storage devices the device under consideration utilises only a single loop mirror and a single SOA as its switch, whilst providing full regenerative capabilities required for long-term storage. I will explain some of the principles and characteristics of the device, which will then be experimentally demonstrated. The device configuration will then be studied and investigated as to its suitability for Hybrid Integrated Technology.

Biological tissues are compositionally and structurally exquisite – a complex network of proteins and cells organised with molecular-precision. Unfortunately, in the absence of an organ transplant or tissue graft, there are no technologies that can completely repair or restore this complex system when it fails. With the hopes of regenerating failing tissue, tissue engineers have developed scaffold structures able to support cell life. As yet, these structures are unable to recreate the complexities of the biological environment, limiting the success of this approach. Nanotechnologies have realised methods to make materials with defined nanoscale properties. Continued research may lead to sophisticated nanobiomaterials, with properties that rival the complexities of biological environments and improve tissue regeneration. To this end, we explored the use of carbon nanotubes (CNTs) within the field of tissue engineering. We investigated the use of 3D CNT scaffolds in bone tissue engineering using strong and porous ceramic scaffold structures coated with CNTs. We abate limitations in previous fabrication methods limiting coating of CNTs throughout porous structures. We demonstrate these surfaces are high quality aligned CNTs, are non toxic and able to support attachment, spreading and proliferation of adipose derived stem cells (ASCs) and human osteoblasts. Following the development of a 3D CNT material, we investigated the potential for using CNTs to create well-defined nanoenvironments capable of regulating cell differentiation. This research is the first report of non-biased quantitative measurement of cell shape during long term differentiation. In contrast to previous techniques, it allows direct measurement of shape rather than that of the underlying substrate. This approach offers novel insights into the relationship between the nanoenvironment, cell shape and cell differentiation. The novel nanomaterials presented in this thesis, demonstrate the potential of nanotechnologies for artificially engineered tissues and organs. Continued research of nanomaterials promises to better recreate the complexities of the biological environment, instructing healthy regenerative processes and promoting tissue function.

Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2013.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 85-97).
Anterior cruciate ligament (ACL) injuries affect over 200,000 Americans yearly, and many occur in young athletes. Current treatment options include tendon autografts and cadaveric allografts. However, these approaches often lead to secondary medical problems, such as donor-site morbidity and immune rejection. Furthermore, in younger patients these grafts fail to grow, leading to additional complications and underlining the need for the development of new approaches that improve the healing and repair of ligaments and tendons. This thesis aims to develop a technique to engineer ACL from autologous mesenchymal stem cells (MSC) and primary ACL fibroblasts using the basic principles of Tissue Engineering. The first part of the thesis characterizes MSCs isolated from tibial bone marrow as an alternative to hip-derived marrow aspirates. The proximity of the tibia to the surgical site of ACL reconstructions makes it a viable source of marrow derived-MSCs for ligament repair, with less stress for the patient and increased flexibility in the operating room. Characterization was performed by fluorescenceactivated cell sorting for MSC-surface markers, and assays to differentiate MSCs towards adipogenic, osteogenic and chondrogenic lineages. The second part of the thesis describes the effects of in vitro co-cultures of ACL fibroblast and MSC on the expression of ligament-associated markers. The goal was to optimize the cell-cell ratio in order to maximize the positive effects of co-cultures on ligament regeneration. Co-cultures of ACL fibroblasts and MSCs were studied for 14 and 28 days in vitro, and the effects assessed with quantitative mRNA expression and immunofluorescence of ligament markers Collagen type I, Collagen type III and Tenascin-C. Finally, based on the enhancing effect observed in co-cultures, the thesis explores a method to regenerate ACL using a three-dimensional polyglyconate scaffold seeded with cell-hydrogel suspensions containing ACL fibroblasts and MSCs. Constructs were analyzed biochemically and by immunofluorescence after 4 weeks in culture with and without mechanical stimulation. Together, our results establish an experimental framework from which a new technique for ACL repair can be developed. The ultimate goal is to foster the design of a one-stage surgical procedure for improved primary ACL augmentation repair that can soon be translated into clinical practice.
by José Antonio Canseco.
Ph.D.in Biomedical Engineering

Infrastructure assets and networks including transport, water and energy systems, are impacted by a range of complex 21st Century challenges from climate change and resource scarcity to rapid technological advances and changing user demands. These challenges highlight that infrastructure assets are integrated components of complex social, ecological and technological systems, rather than siloed technical entities. It is critical therefore that design and engineering responses to such challenges are similarly multi-faceted, adaptive and systems focussed. The relationship between infrastructure and socio-ecological systems is typically twofold. First, infrastructure can negatively impact and disturb living systems, through resource consumption, waste and emissions generation, for example. Secondly, living systems can themselves cause damage to infrastructure, through extreme weather events such as storms, floods and earthquakes, and longer-term trends such as anthropogenic climate change. In this context, infrastructure must become a) more resilient, and capable of withstanding disturbances and impacts generated by living systems, and b) more sustainable, avoiding damage and degradation of those systems. Efforts to enhance both infrastructure sustainability and resilience have typically focused on incremental reductions in damage, however given the scale and complexity of emerging challenges there is increasing demand for innovative responses that move beyond damage reduction towards net positive performance objectives. Here, the intention is to deliver infrastructure and built environments that do not degrade living systems but positively contribute to them. Regenerative design is an emerging discipline that seeks to achieve this. Actionable frameworks and mechanisms for pursuing regenerative design and performance outcomes in infrastructure contexts are critical, though identifying these and making them widely accessible to industry and government has been challenging. A logical source of inspiration is provided by living systems that have developed, tested and refined regenerative design approaches to similarly complex challenges for almost 4 billion years. This approach to learning from living systems is called ‘biomimicry’. This research explores how biomimicry- namely biomimetic design and engineering approaches- can support industry and government in enabling regenerative performance outcomes for infrastructure. Since multi-faceted challenges benefit from multi-faceted research approaches, this research commences with a multidisciplinary review of traditional engineering approaches to resilience and adaptation, and how these approaches may benefit from characteristics of resilience seen in living systems, such as multifunctionality, adaptability, regeneration and real-time sensing and feedback loops. A systematic literature review then provides a first of its kind snapshot of research into biomimetic products, technologies and approaches that can be applied to infrastructure design and engineering. It reveals extensive research into ‘form’ (physical shape and structure) and ‘process’ level solutions, but a clear lack of information regarding ‘system-level’ biomimicry approaches (e.g. patterns and principles) in the built environment. Pursuing regenerative design solutions in response to system-wide challenges, and drawing inspiration from living systems, means it is vital that solutions are also available at the system-level. Though not captured in peer reviewed research, system-level biomimetic design methodologies are indeed being piloted by leaders in industry and government. Hence, challenges and lessons learned were identified by investigating the practical project experiences of six case study projects. Learning from their project, organisation and market level challenges and priorities enabled a distillation of focus areas for future efforts, including 1) improving organisational innovation cultures, 2) enabling emerging market transitions, 3) fostering knowledge sharing and 4) facilitating standardisation through frameworks and standards. This research identifies three pathways for enabling industry and government to readily uptake and mainstream biomimetic design and engineering approaches, informed by those case studies as well as industry workshops undertaken in Australia and the United States of America. The first establishes that infrastructure project governance structures and delivery models can influence the appetite and capacity for innovation in infrastructure design and engineering. Integrated Project Delivery (IPD) models are investigated for this purpose, revealing that the IPD model can support biomimetic innovation by encouraging collaboration across project partners and supply chain. The second pathway is an action framework developed to build the capability of industry, government and academia to implement biomimetic place-based design at the city or regional scale. This pathway allows for upfront biomimetic research and design to benefit multiple projects, assets and networks, improving the feasibility of biomimetic place-based infrastructure design. The third pathway for mainstreaming biomimetic design and engineering is by creating opportunities for government and industry to integrate ‘ecological performance standards’ into environmental impact assessments (EIA) and sustainability rating schemes. This action is enabled through proposed adjustments to the EIA process, as well as the Infrastructure Sustainability (IS) Rating Scheme as an Australian example. This research reveals new insights into the challenges and priorities for government and industry in using biomimetic approaches in infrastructure design and engineering, and establishes pathways for mainstreaming that can guide industry and government in adopting regenerative biomimetic approaches in an infrastructure context. Using biomimicry to create design and engineering solutions inspired by nature can enable solutions that move beyond incremental damage reduction and narrow adaptation approaches, towards infrastructure assets and networks that leverage place-based resilience approaches, adaptive and flexible design, and that generate net positive environmental outcomes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Eng & Built Env
Science, Environment, Engineering and Technology
Full Text

A Spiral Peripheral Neural Interface (SPNI) is an electrode array that has been previously presented as a regenerative neural interface capable of receiving information from, and transmitting information to nerves. The SPNI has previously been proven in concept, however, when stimulating nerves in the device, the electrodes areinsufficiently isolated from each other and stimulations can trigger unwanted neural activity in neighbouring channels of the SPNI. Along with this, neural interfaces generally, suffer from chronic viability problems, due to biological rejection. These issues were addressed in this thesis, by the addition of a PDMS silicone membrane, into the structure of the SPNI. Improvements to the understanding and performance of structural, electrical and biocompatibility aspects of the SPNI are addressed, with the addition of the PDMS film, which is used to electrically seal SPNI channels whilst not hindering conductor integrity. The inclusion ofPDMS also provides a platform which may enable drug delivery. This work dramatically improves SPNI performance whilst providing routes to improved biocompatibility. This thesis addresses the main issues previously presented in the SPNI and brings the device up to a new standard which can once again be tested for its viability in vivo.

Multiple-input multiple-output (MIMO) relaying can increase system throughput, overcome shadowing and expand network coverage more efficiently than its single-antenna counterpart. Non-regenerative strategies, in which the relays apply linear transformation matrices to their received baseband signals before retransmitting them, are favored in many applications due to low processing delays and implementation complexity. In this regard, transceiver design is crucial to fulfilling the great potential of MIMO relay communication systems and we explore this general problem from two different perspectives: coherent combining and adaptation. Within the first perspective, we design linear transceivers for a one-source–multiple-relays–one-destination system in which the source sends information to the destination through multiple parallel relay stations, such that the signals from these relays are coherently combined at the destination to benefit from distributed array gain. Two approaches are proposed: a low-complexity structured hybrid framework and a minimum mean square error (MSE) optimization approach. In the first approach, the non-regenerative MIMO relaying matrix at each relay is generated by cascading two substructures, akin to an equalizer for the backward channel and a precoder for the forward channel. For each of them, we introduced one-dimensional parametric families of candidate matrix transformations. This hybrid framework allows for the classification and comparison of all possible combinations of these substructures, including several previously investigated methods and their generalizations. The design parameters can further be optimized based on individual channel realizations or on channel statistics. This hybrid framework achieves a good balance between performance and complexity. In the second approach, the relaying matrices are designed to minimize the MSE between the transmitted and received signal symbols. Two types of constraints on the transmit power of the relays are considered separately: weighted sum and per-relay power constraints. Under the weighted sum power constraint, we are able to derive a closed-form expression for the optimal solution, by introducing a complex scaling factor at the destination and using Lagrangian duality. Under the per-relay power constraints, we propose a power balancing algorithm that converts the problem into an equivalent one with a weighted sum power constraint. In addition, we investigate the joint design of the MIMO equalizer at the destination and the relaying matrices, using block coordinate descent or steepest descent. The bit-error rate (BER) simulation results demonstrate better performance than previous methods. Within the second perspective, we propose a unified framework for adaptive transceiver optimization for non-regenerative MIMO relay networks. Transceiver designs based on channel state information (CSI) implicitly assume that the underlying wireless channels remain almost constant within each transmission block. This implies that both the channels and the corresponding optimal transceivers evolve gradually across successive blocks. To benefit from this property, we propose a new inter-block adaptive approach based on the minimum MSE criterion, in which the optimum from the previous block is used as the initial search point for the current block. By optimizing the relaying matrices in the first place, we make this adaptation easy to implement by means of iterative algorithms such as the gradient descent. In addition, the proposed framework can accommodate various network topologies by imposing structural constraints on the system model, and leads to new and more efficient algorithms with better performance for certain topologies. We explain in detail how to handle these constraints for different system configurations. Numerical results demonstrate that inter-block adaptation can lead to a significant reduction in computational complexity.
Le relayage multi-entrées multi-sorties (MIMO) permet d'accroître la capacité des systèmes sans fil plus efficacement que sa contrepartie n'utilisant qu'une seule antenne. Les stratégies non-régénératives, dans lesquelles les relais appliquent des matrices de transformation linéaire à leur signaux d'entrée avant de les retransmettre, sont préférées dans de nombre d'applications. La conception de transcepteurs est cruciale afin de pleinement exploiter le grand potentiel qu'offrent les relais MIMO dans les systèmes de communications sans fil. Nous explorons ce problème général à partir de deux perspectives différentes: la combinaison cohérente et l'adaptation. Dans la première perspective, nous concevons des architectures de transcepteur pour un système de type source-simple–relais-multiples–destination-simple (1S-MR-1D) dans lequel la source envoie de l'information à la destination par le biais de plusieurs stations relais en parallèle, de telle sorte que les signaux en provenance des relais se combinent de manière cohérente à la destination. À cette fin, deux approches sont proposées: un schème reposant sur une structure hybride à complexité réduite et une approche d'optimisation basée sur la minimisation de l'erreur quadratique moyenne (MSE). Dans la première approche, les matrices de transformation MIMO non-régénératives utilisées à chacun des relais sont obtenues en cascadant deux sous-structures. Pour chacune de ces sous-structures, nous introduisons une famille paramétrique uni-dimensionnelle de transformations matricielles. Ce schème hybride permet la classification et la comparaison de toutes les combinaisons possibles de ces sous-structures. Les paramètres de conception peuvent de plus être optimisés. Le schème hybride permet d'atteindre un bon équilibre entre la performance et la complexité. Dans la deuxième approche, les matrices de relayage sont conçues de façon à minimiser la MSE entre les symboles transmis et reçus. On considère séparément deux types de contraintes sur la puissance en transmission des relais : la contrainte dite de somme pondérée et la contrainte par relais. Sous la contrainte de somme pondérée, nous développons des expressions mathématiques explicites pour la solution optimale. Sous la contrainte de puissance par relais, nous proposons un algorithme de balancement qui permet de convertir le problème d'optimisation en un problème équivalent avec contrainte de type somme pondérée. De plus, nous étudions le problème de la conception jointe de l'égalisateur MIMO à la destination et des matrices de relais. Les résultats de simulation démontrent une performance supérieure à celle de méthodes existantes. Dans la deuxième perspective, nous proposons un cadre unifié d'optimisation des transcepteurs adaptatifs pour les réseaux de relais MIMO non-régénératifs. La conception de transmetteur basée sur l'information de l'état du canal (CSI) suppose implicitement que les canaux sans fil demeurent constants durant chaque bloc de transmission. Cela implique que les canaux et les transcepteurs optimaux correspondants évoluent graduellement au passage des blocs. Afin de bénéficier de cette propriété, nous proposons une nouvelle approche d'adaptation inter-bloc basée sur le critère de minimisation de la MSE. Dans cette approche, la solution obtenue du bloc précédent est utilisée comme point de départ dans la recherche d'une solution optimale pour le bloc actuel. Fortuitement, il est possible d'optimiser les matrices de relayage de façon analytique en premier lieu, ce qui facilite grandement l'adaptation des paramètres restants au moyen d'algorithmes itératifs tels que celui de la descente de gradient. De plus, le cadre d'optimisation que nous proposons peut être adapté à des topologies de réseau variées par l'imposition de contraintes structurelles sur le modèle. Les résultats de simulations numériques démontrent que l'adaptation inter-bloc peut conduire à une réduction importante de la complexité numérique.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1988, and (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1988.
Includes bibliographical references.
by Keith Aaron Tabor.
M.S.

Anterior cruciate ligament (ACL) ruptures and tears are significant orthopedic problems that result in discomfort and limited mobility. Fully functional tissue engineered ligament replacements are promising alternatives to current graft choices for repair of ACL disruptions. The cell-based approach to construct engineered ligament grafts presented herein involves the culture of mesenchymal stem cells (MSC) on biodegradable, fibrous polymeric scaffolds to promote tissue formation. Multipotent MSCs are advantageous because of their in vitro proliferative capacity and ease of harvest; however; the promotion of MSC differentiation into mature fibroblasts and subsequent extracellular matrix (ECM) development is unknown. The proposed studies utilized three complementary methods to promote differentiation of MSCs: scaffold architecture, mechanical stretch and over-expression of the transcription factor, scleraxis. First, elastomeric scaffolds were fabricated by electrospinning a segmented poly(esterurethane urea) with variations in fiber diameter and fiber alignment. Primary mesenchymal stem cells and the mesenchymal stem cell line, C3H10T1/2, were seeded on these scaffolds and assumed spindle-shaped morphologies and oriented with the direction of fiber alignment. Fiber diameter affected cellular responses, including the expression of ECM genes (e.g. collagen type 1 and decorin) which were elevated on smaller mean fiber diameter scaffolds initially. However, scleraxis gene expression was greatest on larger mean fiber diameter scaffolds at the end of two weeks. Second, cyclic stretch was applied to C3H10T1/2 cells on semi-aligned scaffolds using a novel bioreactor. Cell attachment was verified during and after the application of mechanical stress by confocal microscopy. Cyclic stretch induced cells to assume a highly elongated morphology; however ECM gene expression changes were moderate. Third, forced constitutive expression of scleraxis was accomplished by nucleofection of C3H10T1/2 cells. Transient mRNA expression, accumulation of the gene product in the cell nucleus, and cell death were observed. Future work will seek to refine the experimental methods, including the development and testing of an inducible scleraxis transgene and the application of longer periods of mechanical stimulation. Finally, these complementary approaches may be combined to further extend this work in pursuit of directed differentiation of stem cells and the ensuing generation of a robust tissue graft.
Ph. D.

Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.

This thesis describes the development and utilisation of a self-reporting scaffold to improve current monitoring methods of the cellular microenvironment. In vitro tissue models hold a lot of promise for regenerative medicine and tissue engineering. However, many models lack the ability to non-invasively monitor in situ cellular responses in a physiologically relevant environment. By development of electrospun self-reporting scaffolds and incorporation of flow culture conditions, this limitation can be overcome. Electrospun matrices have been shown to mimic the structural architecture of the native extracellular matrix, whilst flow conditions have been shown to regulate cellular processes, and enhance mass transport and nutrient exchange throughout polymeric scaffolds. Here we show the development of optically transparent self-reporting electrospun scaffolds that incorporate ratiometric pH-sensitive nanosensors and respond to biological and mechanical cues of the native extracellular matrix through exposure to shear stress. Optically transparent self-reporting scaffolds were fabricated by directly electrospinning pH responsive, ratiometric nanosensors within a gelatin biopolymer matrix. The sensors consist of a porous polyacrylamide matrix which encapsulates pH-sensitive fluorophores that exhibit an additive fluorescent response across the full physiological range between pH 3-8, and a pH-insensitive reference fluorophore. The self-reporting scaffold was able to support cell growth whilst being able to simultaneously monitor local pH changes in real time. A Quasi-Vivo® bioreactor system was also used to generate a flow of cell culture medium and expose cell-seeded scaffolds to a continual shear stress. This novel diagnostic scaffold and the use of flow conditions can help simulate enhance the understanding of in vitro conditions, and generate advanced simulations in vivo to facilitate tissue engineering and regenerative medicine applications.

Repair of large bone defects remains a clinical challenge for orthopedic surgeons. Current treatment strategies such as autograft and allograft are limited by the amount of available tissue in the case of the former, and failure of revascularization effecting engraftment in the case of the latter. Tissue engineering offers an alternative approach to this challenging clinical problem. The general principle of tissue engineering for bone regeneration prescribes delivery of osteoinductive factors to induce an endogenous response within the host to repair a defect that will not normally heal. One such tissue engineering approach is cell based therapy and this is attractive in the cases of patients with a lack of endogenous osteoprogenitors cells due to volumetric loss of tissue/ageing. Stem cell therapy has emerged as a possible alternative to current treatment modalities, however many challenges to clinical translation remain. Central to these challenges for bone tissue engineering are lingering questions of which cells to use and how to effectively deliver those cells. The goal of this thesis was to elucidate more effective ways to enhance bone repair utilizing adult stem cells. First, we investigated adipose derived stem cells (ADSCs) as a viable cell source for bone tissue engineering. Upon isolation, adipose derived stem cells are a heterogeneous population of multipotent cells predisposed to adipogenic differentiation. We developed an enrichment protocol that demonstrated the osteogenic potential of ADSCs can be enhanced in a dose dependent manner with resveratrol, which had been demonstrated to up-regulate Runx-2 expression. This enrichment strategy produced an effective method to enhance the osteogenic potential of ADSCs while avoiding cell sorting and gene therapy techniques, thus bypassing the use of xenogenic factors to obtain an enriched source of osteoprogenitor cells. This protocol was also used to investigate differences between human and rat ADSCs and demonstrated that rat ADSCs have a higher osteogenic potential than human ADSCs in vitro. The second major thrust of this thesis was to develop an injectable hydrogel system to facilitate bone formation in vivo. Both a synthetic and a naturally based polymer system was investigated, the results of which demonstrated that the naturally based alginate hydrogel was a more effective vehicle for both cell viability in vitro and bone formation in vivo. Our results also demonstrated that despite the ability to increase the osteogenic potential of ADSCs in vitro with resveratrol treatment, this was insufficient to induce bone formation in vivo. However, the inclusion of bone marrow mesenchymal stem cells (BMMSCs) in BMP-2 functionalized alginate hydrogels resulted in significantly greater mineralization than acellular hydrogels. Finally, the effect of timing of delivery of therapeutics to a non-healing segmental bone defect in the femur was investigated. We hypothesized that delivery of biologics after the initial inflammation response caused by injury to the host tissue would result in greater regeneration of tissue in terms of newly formed bone. Contrary to our initial hypothesis, these experiments demonstrated that delayed implantation of therapeutics has a detrimental effect on the overall healing response. It was, however, demonstrated that the inclusion of BMMSCs results in greater bone volume regenerated in the defect site over acellular hydrogels. In conclusion, this work has rigorously investigated the use of adipose derived stem cells for bone tissue engineering, and further produced an injectable hydrogel system for stem cell based bone tissue engineering. This work also demonstrated that the inclusion of adult stem cells, specifically BMMSCs, can enhance the regeneration response in a non-healing bone defect model relative to acellular hydrogel.

Bioengineering
Ph.D.
Electrospinning of nanofibrous mats and scaffolds enables generation of scaffolding that is not only highly porous, but also has a structure that essentially mimics the natural basement membrane. As a result, the method has proliferated extensively, and is commonly used for diverse applications such as water filtration or tissue engineering, the latter of which may involve the use of natural or synthetic materials. Common laboratory scale electrospinning setups can be built inexpensively with merely a syringe pump, a high voltage supply, and an aluminum foil target. These systems, however, are limited to flat target surface geometries that span several centimeters. While a scaffold can be cut or folded to conform to a bone or other biological surface, spinning directly onto a surface with significant peaks and troughs results in poor fiber uniformity. Furthermore, if an alteration of fiber properties is preferred, the high voltage setup limits user access and customization of parameters during the spinning period. Finally, control of the electric field is compromised by the proximity of grounded electrical components. As its first aim, this project develops a robotic control system to enable custom coatings of arbitrary surfaces. By augmenting the traditional electrospinning system with a three-dimensional robotic control system, electric field focusing fibers, and additional aerodynamic forces terms ‘electroblowing’, the device can be produced across targets with strong topographic anisotropy. The second aim continues to enhance these attributes with biocompatible soy based scaffolds. Craniofacial implants are often complex in geometry, and conformal bandages are particularly hard to produce in these areas. Soy based scaffolds will be produced for 3D-printed replicas of these situations. Finally, the methods developed across this aim enables the development and use of a handheld electrospinning system that combines a coaxial high velocity air flow with the high voltage spinning element to reduce effects of operator error. The final goal of the thesis is to test whether fiber control successfully reduces effects of fiber anisotropy in vitro and to use the enhanced fiber control mechanisms to produce scaffolds with significant anisotropy, depositing aligned fibers at a target point to eventually enable generation of scaffolds with programmable variable spatial alignment similar to tendon. When completed, the systems described will enable custom production of coatings or scaffolds for functionality as scaffolding on medically relevant surfaces. Specifically, this means first, that scaffolds can be used with confidence to improve fixation even of non-cylindrical implants and enhance local tissue integration, and second, that implants can be customized with areas of ‘guidance’ fibers or local drug depots to either promote regeneration and population by surrounding tissue or mimic natural anisotropic cues necessary for mechanical or biological functionality.
Temple University--Theses

Recoverable energy in vehicle suspension systems has attracted intensive attention in recent years for the improvement of vibration suppression performance and the reduction of energy dissipation. Various design concepts and structures of regenerative suspensions have been presented and investigated to recover the energy of linear motion and vibration between the vehicle body and chassis from road disturbances. These studies concentrate on the energy conversion from kinetic energy to electricity. Although a large number of concepts and models have been proposed and evaluated to regenerate power for reuse, the previous simulation works have used significantly simplified models without considering parameter uncertainties and system losses. In addition, experimental works are too simple to support for modelling optimisation. To advance the technology, a regenerative hydraulic shock absorber is investigated rigorously by examining the system at various developing stages including modelling all hydraulic, mechanical, and electromagnetic processes, simulating its behaviours, identifying its uncertain parameters/variables, fabricating a prototype of a commonly used shock absorber, testing its desirable performance and evaluating its on-road usability, which has given an accurate understanding of dynamic behaviours and power regeneration of a regenerative hydraulic shock absorber system. Based on the configuration of the prototype, a comprehensive mathematical model is developed for the regenerative hydraulic shock absorber system. The various losses and nonlinearity have been taken into account in modelling hydraulic, mechanical, and electromagnetic processes, which allow more detailed influences and agreeable predictions with the experimental work to be obtained. The introduction of the gas-charged hydraulic accumulator into the system has been explored in both modelling and testing to provide power smoothing in an attempt to give a more stable recoverable power. Model parameter identifications and refinements based on online data are systemically investigated. It has found that the pressures, rotation speeds and electrical outputs, which are readily available in the system, are sufficient to determine and refine uncertain model parameters such as the voltage constant coefficient, torque constant coefficient, generator internal resistance and rotational friction torque using a common least square method. The developed experimental rig and measurement systems for the study of regenerative hydraulic shock absorbers are designed and built. The variations in motor pressure and shaft speed under different excitations are evaluated, and also voltage output and recoverable power at different electrical loads are investigated. Additionally, the experimental work is not only used to validate the predicted results comprehensively, but also to offer a practical evaluation method for the system under various operating conditions. In particular, the system using piston-rod dimensions of 50-30mm achieves recoverable power of 260W with an efficiency of around 40% under sinusoidal excitation of 1Hz frequency and 25mm amplitude. Additionally, control strategies and their realisation on a general purpose PC computer are developed based on constant voltage, current and resistance schemes to carry out the investigation of the system performances, which allows it to be fully evaluated upon the compromise between the damping behaviour and power regeneration performance for different road conditions. Furthermore, the simulation of the entire system and parameter computations are all realised on the Matlab platform, which provides sufficient flexibility to take into account more influence factors for accurate and detailed analysis and thus can be an effective mathematical tool for further development research in this direction such as the optimisation of the structures, control strategies and system integrations.

This thesis presents the design and the fabrication process of a three-dimensional (3D) neural interface consisting of a bundle of parallel micro-channels with (100μmx100μm) cross-sectional area and embedded micro-electrodes. This is a regenerative implant that is able to stimulate and record extracellular neural signals in the peripheral nervous system as demonstrated by the \(in-vivo\) experiments conducted in collaboration as part of this project. These implants have the potential to be developed into long-term neural interfaces capable of extracting neural signals from stumps of severed peripheral nerves to use as control inputs for muscles simulators or artificial limbs for amputees. The skeleton of the device is entirely made of flexible polyimide films. Gold micro-electrodes and micro-channels of photosensitive polyimide are patterned directly on polyimide substrates. After fabrication, the 2D electrode micro-channel array is rolled into a 3D structure forming concentric rolls of closed micro-channel arrays with a Swiss-roll like arrangement. Microflex Interconnection technique (MFI) was incorporated successfully into the implant. The performance of the implant microelectrodes was characterised \(in-vitro\) through impedance spectroscopy and \(in-vivo\) via implantation in animals for three months. The ability of the electrodes to stimulate and capture action potentials from regenerated tissue was also assessed.

High performance dc servo-drives are often supplied from four-quadrant choppers to ensure low armature current ripple and large control loop bandwidths. In order to allow for continued regenerative braking, the dc link voltage is regulated within limits and the energy produced during braking is dissipated in resistances.
In the proposed scheme the energy produced during regeneration is re-injected into the ac mains supply. The scheme consists of adding to each diode of the front end rectifier a transistor capable of conducting the reverse current, and suppressing the dc link capacitor.
The performance of the complete converter with a single phase ac mains is analysed in terms of operation in both motoring and braking modes. The advantages of the scheme are presented and its characteristics are compared to the standard chopper configuration and to the conventional phase-controlled converter systems. It is shown that the system exhibits high power factor and efficiency and is very compact.

Nowadays, there are approximately 10 million people worldwide with visual impairment due to corneal diseases. Currently, the main therapeutic solution is the transplant of a donor's cornea. The great majority of transplants is due to some failure in the inner layer of the cornea, which is called the corneal endothelium and this is mainly related with the inability of this layer to regenerate in vivo. However, transplants present several limitations such as the low number of healthy donors or immunological rejection by the patient. In order to overcome these problems, several researchers have focused in culturing corneal endothelial cells (CEC) to subsequently replace non-functional CEC. However, cell therapy is still very recent and still presents a series of drawbacks. For instance, using animal CEC or cells from other patients has shown to lead into immunological rejection. In order to avoid this, it is possible to use stem cells from the same patient, which have the ability to differentiate into many cell types, including the corneal endothelium. Currently, the stem cells used to regenerate CEC are mainly pluripotent stem cells, either embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), which are derived from adult cells. Despite their great potential for treating diseases, these types of stem cells present major limitations such as the risk of teratoma formation. In addition, they present other disadvantages such as ethical problems associated with the use of ESCs, safety problems related to iPSC since they requires the use of virus for their production hence limiting its clinical application. For this reason, and in order to solve the current problems in the regeneration of corneal endothelium, this thesis project uses dental pulp stem cells (DPSC) for the formation of CEC. DPSC are an accessible source derived from the same patient, avoiding possible future problems of rejection. In addition, the use of DPSC avoids the ethical and security problems associated with ESC and iPSC. Furthermore, DPSC and CEC have the same embryological origin, as they both arise from neural crest stem cells. In fact, DPSC express neural crest stem cells markers, which facilitates their differentiation into neural crest stem cells (NCSC), which is an intermediate step for the formation of CEC. Therefore, this thesis project uses a two-step protocol, where DPSC are differentiated into NCSC and, subsequently, NCSC are derived into CEC. Because the use of cell therapies alone may present limited cell viability once it is injected, the field of tissue engineering is a new discipline that has appeared to overcome this limitation. Tissue engineering combines the use of cells, biomaterials and biological molecules. It has been demonstrated that the use of different topographies in cell culture modulates cell behavior, and may have an effect on their functionality, cell distribution or cell size. Therefore, this thesis project applies tissue engineering as another strategy for the generation of functional CEC with its characteristic phenotype and morphology. For doing this, we have mimicked the natural CEC environment by cultivating the cells on substrates with different curvatures, composition or topographies that are able to mimic those of the human eye. In conclusion, this thesis project proposes the use of bioengineering, by differentiating CEC from stem cells derived from the patient and the use of biomaterials with different topographies and curvatures, for the regeneration of corneal endothelium.

Bone is a highly organised and specialised connective tissue that provides a rigid, protective and supporting framework to the body. In addition to this, bone is unique in its capacity to self-regenerate without the formation of a fibrotic scar. Despite its natural healing potential, bone is not always able to repair large defects, which can result in permanent bone loss and fracture non-unions. Consequently, interventions such as bone grafting are required to replace damaged or diseased bone, accounting for more than two million grafted bones in the world annually. Currently, autografts are still qualified as the gold standard technique, but this option is not exempt of complications such as infections and donor site morbidity. Advanced therapies (AT), particularly regenerative medicine (RM) and tissue engineering (TE) approaches, provide valuable tools with broad applicability in the orthopaedic field with the aim of achieving bone regeneration. This PhD project was developed within the RM field aiming to optimise the formulation of tissue engineering products (TEPs) composed of mesenchymal stromal cells (MSCs) for bone regeneration. To date, MSC-based therapies have been demonstrated safe and some initial signs of efficacy have already been found in several clinical indications; that is why current major challenges rely on improving the efficacy of such therapies by modifying formulations paying special attention to the tissue source of MSCs as well as to the non-cellular components of the final TEPs. The proposal reported in this PhD project is based on the use of MSCs derived either from bone marrow (BM) or the Wharton’s jelly (WJ) of the umbilical cord (UC) as the osteogenic component of the TEP, decellularised bony particles providing osteoinductive and osteoconductive cues and a hydrogel made of fibrin which confers the ability of adapting to the architecture of each particular defect. The investigation has been performed in vitro and in vivo in an ectopic mice model (addressed in CHAPTER IV) and subsequently in two orthotopic ovine models (addressed in CHAPTER III and CHAPTER V) demonstrating an excellent safety profile and signs of efficacy. The new BM-derived MSCs-based clinical grade formulation developed in this work resulted feasible, effective and efficiently adapted to the architecture of simulated cylindrical bone defects. On the other hand, this work is a milestone in the non-clinical development of WJ-MSCs-derived TEPs prior to use in patients. Nonetheless, further investigations in order to trigger the osteogenic commitment of WJ-derived MSCs for specific bone regeneration indications are required. In addition, the outcomes relating to the injectable bone formulation make it an attractive alternative to be considered in future TE approaches regarding three dimensional (3D) bioprinting, as a potential MSC-based bioink.
El hueso es un tipo de tejido conjuntivo altamente especializado y organizado que proporciona una estructura de soporte rígida y protectora. Además, el hueso es único en su capacidad de autoregeneración sin la formación de una cicatriz fibrótica. A pesar de su potencial regenerador natural, el hueso no es siempre capaz de reparar grandes defectos por sí solo, lo que puede resultar en pérdidas óseas permanentes o en la aparición de pseudoartrosis. Por consiguiente, se requieren intervenciones quirúrgicas para la aplicación de injertos con la finalidad de reemplazar hueso dañado o enfermo. Esto se traduce en la implantación de más de dos millones de injertos óseos anuales en el mundo. Actualmente, los autoinjertos siguen siendo la técnica quirúrgica estándar, pero no están exentos de complicaciones, tales como infecciones o morbilidad asociada a la zona de extracción donante. Las terapias avanzadas (AT), particularmente las aproximaciones dentro de la medicina regenerativa (RM) y la ingeniería de tejidos (TE), ofrecen herramientas valiosas con amplia aplicabilidad en el mundo de la ortopedia con el objetivo de lograr regenerar hueso. Esta tesis doctoral se ha desarrollado dentro del campo de la RM con el objetivo de optimizar la formulación de productos de ingeniería de tejidos (TEPs) compuestos por células mesenquimales estromales (MSCs) con la finalidad de regenerar hueso. Hasta la fecha, se ha acumulado amplia experiencia, tanto preclínica como clínica, demostrando la seguridad e indicios de eficacia de las terapias basadas en el uso de MSCs para diversas indicaciones. Por este motivo, los principales retos en la actualidad se centran en mejorar la eficacia de dichos productos modificando sus formulaciones y prestándole especial atención tanto al tejido de aislamiento de las MSCs como a los componentes no celulares de los TEPs. La propuesta presentada en esta tesis doctoral está basada en MSCs derivadas de médula ósea (BM) o de gelatina de Wharton (WJ) del cordón umbilical (UC) como componente osteogénico, partículas de hueso descelularizadas que aportan las propiedades osteoinductoras y osteoconductoras y un hidrogel de fibrina que confiere la habilidad de adaptarse a la arquitectura de cada defecto en particular. La investigación se ha realizado tanto in vitro como in vivo en un modelo ectópico en ratón (abordado en el CHAPTER IV) y subsecuentemente, en dos modelos ortotópicos en oveja (abordado en el CHAPTER III y en el CHAPTER V) demostrando seguridad y signos de eficacia. La nueva formulación de grado clínico basada en MSCs derivadas de BM resultó ser factible, eficaz y eficiente adaptándose a la arquitectura de los defectos cilíndricos simulados. Por otro lado, este trabajo es un hito en el desarrollo no clínico de los TEPs basados en MSCs derivadas de WJ antes de su aplicación en pacientes. No obstante, se requieren más estudios con el objetivo de desencadenar la rápida diferenciación hacia linaje osteogénico de las MSCs derivadas de WJ en indicaciones específicas de regeneración ósea. Además, los resultados relacionados con la formulación de hueso inyectable la convierten en una alternativa atractiva para ser considerada en futuras aproximaciones de TE relacionadas con la bioimpresión en tres dimensiones (3D), como una potencial biotinta basada en MSC.

Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.

Vat photopolymerization (VP) is an additive manufacturing (AM) technology that permits the fabrication of parts with complex geometries and feature sizes as small as a few microns. These attributes make VP an attractive option for the fabrication of scaffolds for tissue engineering. However, there are few printable materials with low cytotoxicity that encourage cellular adhesion. In addition, these resins are not readily available and must be synthesized. A novel resin based on 2-acrylamido-2-methyl-1-propanesulfonic acid (NaAMPS) and poly(ethylene glycol) diacrylate (PEGDA) was formulated and printed using VP. The mechanical properties, water content, and high fidelity of the scaffold indicated promise for use in tissue engineering applications. Murine fibroblasts were observed to successfully adhere and proliferate on the scaffolds. The growth, migration, and differentiation of a cell is known to dependent heavily on its microenvironment. In engineered constructs, much of this microenvironment is provided by the tissue scaffold. The physical environment results from the scaffold's geometrical features, including pore shape and size, porosity, and overall dimensions. Each of these parameters are known to affect cell viability and proliferation, but due to the difficulty of isolating each parameter when using scaffold fabrication techniques such as porogen leaching and gas foaming, conflicting results have been reported. Scaffolds with pore sizes ranging from 200 to 600 μm were fabricated and seeded with murine fibroblasts. Other geometric parameters (e.g., pore shape) remained consistent between scaffold designs. Inhomogeneous cell distributions and fewer total cells were observed in scaffolds with smaller pore sizes (200-400 μm). Scaffolds with larger pores had higher cell densities that were homogeneously distributed. These data suggest that tissue scaffolds intended to promote fibroblast proliferation should be designed to have pore at least 500 μm in diameter. Techniques developed for selective placement of dissimilar materials within a single VP scaffold enabled spatial control over cellular adhesion and proliferation. The multi-material scaffolds were fabricated using an unmodified and commercially available VP system. The material preferences of murine fibroblasts which resulted in their inhomogeneous distribution within multi-material scaffolds were confirmed with multiple resins and geometries. These results suggest that multi-material tissue scaffolds fabricated with VP could enable multiscale organization of cells and material into engineered constructs that would mimic the function of native tissue.
Doctor of Philosophy
Vat photopolymerization (VP) is a 3D printing (or additive manufacturing) technology that is capable of fabricating parts with complex geometries with very high resolution. These features make VP an attractive option for the fabrication of scaffolds that have applications in tissue engineering. However, there are few printable materials that are biocompatible and allow cells attachment. In addition, those that have been reported cannot be obtained commercially and their synthesis requires substantial resources and expertise. A novel resin composition formulated from commercially available components was developed, characterized, and printed. Scaffolds were printed with high fidelity. The scaffolds had mechanical properties and water contents that suggested they might be suitable for use in tissue engineering. Fibroblast cells were seeded on the scaffolds and successfully adhered and proliferated on the scaffolds. The growth, migration, and differentiation of cells is influenced by the environmental stimuli they experience. In engineered constructs, the scaffold provides many of stimuli. The geometrical features of scaffolds, including how porous they are, the size and shape of their pores, and their overall size are known to affect cell growth. However, scaffolds that have a variety of pore sizes but identical pore shapes, porosities, and other geometric parameters cannot be fabricated with techniques such as porogen leaching and gas foaming. This has resulted in conflicting reports of optimal pore sizes. In this work, several scaffolds with identical pore shapes and porosities but pore sizes ranging from 200 μm to 600 μm were designed and printed using VP. After seeding with cells, scaffolds with large pores (500-600 μm) had a large number of evenly distributed cells while smaller pores resulted in fewer cells that were unevenly distributed. These results suggest that larger pore sizes are most beneficial for culturing fibroblasts. Multi-material tissue scaffolds were fabricated with VP by selectively photocuring two materials into a single part. The scaffolds, which were printed on an unmodified and commercially available VP system, were seeded with cells. The cells were observed to have attached and grown in much larger numbers in certain regions of the scaffolds which corresponded to regions built from a particular resin. By selectively patterning more than one material in the scaffold, cells could be directed towards certain regions and away from others. The ability to control the location of cells suggests that these printing techniques could be used to organize cells and materials in complex ways reminiscent of native tissue. The organization of these cells might then allow the engineered construct to mimic the function of a native tissue.

Severe traumatic injuries and surgical procedures like tumor resection often create peripheral nerve gaps, accounting for over 250,000 injuries in the US annually. The clinical "gold standard" for bridging peripheral nerve gaps is autografts, with which 40-50% of patients regain useful function. However, issues including their limited availability and collateral damage at the donor site limit the effectiveness and use of autografts. Therefore, it is critical to develop alternative bioengineered approaches that match or exceed autograft performance. With the use of guidance channels, the endogenous regeneration process spontaneously occurs when successful bridging of short gaps (< 10mm) occurs, but fails to occur in the bridging of longer gaps (≥15mm). Several bioengineered strategies are currently being explored to bridge these critical size nerve gaps. Other labs and ours have shown how filler materials that provide topographical cues within the nerve guides are able to enhance nerve growth and bridge critical length gaps in rats. However, the mechanism by which intra-luminal fillers enhance nerve regeneration has not been explored. The main goal of this dissertation was to explore the interplay between intra-luminal scaffolds and orchestrated events of provisional fibrin matrix formation, glial cell infiltration, ECM deposition and remodeling, and axonal infiltration - a sequence we term the 'regenerative' sequence. We hypothesized that the mechanism by which thin films with topographical cues enhance regeneration is by serving as physical 'organizing templates' for Schwann cell infiltration, Schwann cell orientation, extra-cellular matrix deposition/organization and axon infiltration. We demonstrate that aligned topographical cues mediate their effects to the neuronal cells through optimizing fibronectin adsorption in vitro. We also demonstrate that aligned electrospun thin films are able to enhance bridging of a critical length nerve gap in vivo by stabilizing the provisional matrix, creating a pro-inflammatory environment and influencing the maturation of the regenerating cable leading to faster functional recovery compared to smooth films and random fibers. This research will advance our understanding of the mechanisms of peripheral nerve regeneration, and help develops technologies that are likely to improve clinical outcomes after peripheral nerve injury.

The interface between tendon/ligament and bone tissue is a complex transition of biochemical, cellular, and mechanical properties. Investigating computational and tissue engineering models that imitate aspects of this interface may supply critical design parameters for designing future tissue replacements to promote increased biochemical and mechanical integration between tendon/ligament and bone. Strategies for modeling this tissue have typically focused on the development of heterogeneous structures to create gradients or multiphasic materials that mimic aspects of the transition. However, further work is required to elucidate the role of specific mechanical and material stimuli in recapitulating features of the tendon/ligament-bone insertion. In particular, in constructs that exhibit variation in both mechanical and biochemical properties, the interplay of mechanical, material, and chemical signals can complicate understanding of the particular factors at work in interface formation. Thus, the overall goal of this dissertation was to provide insight into the role of mechanical strain and scaffold degradability on cell behavior within heterogeneous biomaterials. Specifically, a method for determining cell vertical position within a degradable gel through a laminated interface was developed. A computational model was created to examine possible variation in local mechanical strain due to heterogeneity in mechanical properties and different interface geometries. Finally, the influence of biomaterial degradability on changes in encapsulated human mesenchymal stem cell morphology under response to cyclic mechanical strain was explored. Together, these studies provide insight into mechanical and material design considerations when devising tissue engineering strategies to regenerate the tendon/ligament-bone interface.

Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

Abstract Drug delivery system (DDS) technology is particularly promising to improve the in vivo efficiency of active molecules. Moreover, it is possible to stabilize and prolong the half life at biologically active molecules, thus prolonging the in vitro activity. DDS's can be used either for the delivery of anticancer drug but also for the controlled release of growth factors essential for the tissue engineering. In this work DDS’s have been investigate to develop new targeted anticancer drug or to control the release of growth factor for tissue engineering. In the first case polymer conjugates were chosen while in the second microspheres were used. PEG conjugates Anti-cancer drugs are very active molecules but they present limits that often prevent their success in chemotherapy. Common problems are a low half-life due to rapid kidney clearance, rapid inactivation by metabolic enzymes and low selectivity towards cancer cells and often a low solubility in water, these causing severe side effects. To overcome this problems, polymeric conjugates were prepared by linking an anti-cancer drug to a polymer carrier. Polymeric conjugation improves the drug pharmacokinetic profiles by reducing drug clearance. Furthermore, a tumor targeting can be reached by two mechanisms: the first a passive accumulation into tumour tissue, known as the EPR (Enhanced Permeability and Retention) effect and the second an active targeting when a targeting molecule also is coupled to the conjugate. In this work an heterobifunctional poly-(ethylene glycol) was coupled to both epirubicin (EPI), an anti-cancer drug, and to folic acid (FOL) as targeting residue. The biological activity of the derivate FOL-PEG-EPI was studied in two different culture systems; the classic bi-dimensional (2D) system and the tri-dimensional (3D) system using Puramatrix hydrogel. The last should recreate an environment similar to the in vivo situation. The cytotoxicity activity studies were carried out on the following cell lines; HT-29, expressing a normal level of folic acid membrane receptor (FR), MCF-7, medium FR expression and KB-31cells over-expressing FR. The FOL-PEG-EPI cytotoxicity study, showed higher toxicity in KB-31 cells than MCF-7 and HT-29 in both 2D and 3D cell culture systems. Moreover, the use of the 3D culture system, displayed clearly that FOL-PEG-EPI had selective activity on cells over-expressing the folic acid receptor (KB- 31) compared to HT-29cells where to obtain the IC50 was used a conjugate concentration 3 fold higher than the maximum one permitted in clinic. The uptake of conjugates and epirubicin were studied by flow cytometry, and confocal microscopy. In the first case the cytometry showed the fluorescence signal inside the cells for both FOL-PEG-EPI and epirubicine alone. Also, the confocal analysis have confirmed the internalization, displayed the epirubicin in the nuclei and the conjugate in perinuclear side. Microspheres Tissue engineering is based on various disciplines such as medicine, biology, engineering and chemistry fused to the common aim to obtain or replace organs, or parts of organs in the human body. In general, a construct of tissue engineering is formed by the cellular component and a basic structure with function of support. The cells need to be stimulate by proper growth factor to generate a functional tissue. When the solution of growth factors is injected into the site for regeneration, the biological effect is not always optimal, because the biologically active substances are spread away from the site of action very quickly, or because they cannot get to the targeting site. It is therefore essential to develop a technology that stabilizes the administration and the answer can be a DDS. Therefore, the second part of this study was focused on tissue engineering of bone tissue. The research involved the use a the drug delivery system for the controlled release of TAT-OP1 protein, which stimulates osteogenic differentiation. Osteogenic protein-1 (OP-1 or BMP-7) is a member of Bone Morfogenic Proteins’s family (BMPs). BMPs are a group of multi-functional growth factors belonging to the transforming growth factor β (TGF-β) superfamily. They are implicated in a variety of functions such as the formation of cartilage and bone, and the development of non-osteogenic tissues. BMPs are secreted as a precursor approximately four times longer than the mature form and share a C-terminal distinctive pattern (..C…CXGXC…CC…CXCX..) containing seven cysteines which are the active region of the proteins. In this study we used a recombinant fusion protein called TAT-OP1 which includes a TAT sequence, an Arg rich peptide, derived from a HIV protein, which allows the internalization. The construct TAT-OP1 has 162 amino acids starting with an N-terminal 6His-tag followed by the TAT sequence, a peptidase specific cleavage site (spanning 6 AA) and the C-terminal OP-1 domain (126 AA) containing the cysteines motif. When this type of bioactive molecules is injected directly on the action site, it undergo a rapid inactivation and dilution effect, therefore this is the limit for in vivo use. To avoid this problem, the protein was encapsulated in polylactidecoglycolide (PLGA) microspheres. The bioactive molecules released from microspheres can be easily modulated by setting the formulation parameters and production technique. The spray drying technique was used to obtain the TAT-OP1 microspheres. The microspheres release of the TAT-OP1 over a period of 7 days was 98% and the encapsulation efficiency was 35%. The size measurement by SEM was 0.2-2 μm. The biological activity study on TAT-OP1 microspheres was conducted using pre-osteoblast MC3T3-E1 cell line at two different concentrations, 200nM and 27 nM. After the 7 and 14 days of treatment the cultures showed matrix mineralization and the assay testing for the alkaline phosphatase was positive. Also the presence of characteristic osteogenic markers, such as osteopontin and osteocalcin, was verified by immunofluorescence. These positive results led us to evaluate the biological activity of TAT-OP1 microspheres in a tri-dimensional culture system on cells isolated from umbilical cord blood (UCBMSC). The 3D model was made by using synthetic Puramatrix TM Hydrogel which is able to mimic the natural microenvironment. Following encapsulation of the TAT-OP1 microspheres or the free TAT-OP1 into Puramatrix Hydrogel TM, the cellular response to TAT-OP1 stimulation was evaluated using transmission electron microscopy (TEM) analysis to detect the production of bone-matrix. After 27 days of stimulation with TAT-OP1 loaded microspheres(200 nM), partially aggregated microfibrils were observed around the cells. Calcification deposit and hydroxyapatite crystals were detected only in the cultures treated with TAT-OP1 PLGA microspheres (200nM) controlled release system. Therefore the controlled release of TAT-OP1 from PLGA microspheres was verified to increase the stimulation effectiveness. Future investigations will be directed to further confirm the suitability of this approach to improve the in vitro osteogenic differentiation and the biological activity of TAT-OP1 for an eventual clinical application in the field of bone tissue engineering.
Riassunto I sistemi di drug delivery (DDSs) rappresentano una tecnologia particolarmente promettente per migliorare l'efficacia in vivo e in vitro di molecole biologicamente attive con l’obiettivo di circoscriverne l’effetto su una determinata tipologia di cellule, migliorarne l’efficacia, prolungarne il periodo di emivita e ridurre la tossicità di una terapia. In questo lavoro sono stati studiati due modelli di Drug Delivery: il primo riguarda lo sviluppo di nuovi farmaci antitumorali selettivi mediante un coniugato polimerico, mentre il secondo modello, che trova applicazione nell’ambito dell’ingegneria tissutale, riguarda il rilascio controllato di fattori di crescita mediante microsfere. PEG coniugato I problemi più comuni riguardanti i farmaci anti-tumorali possono essere dovuti ad un tempo di emivita basso a causa di clearance renale rapida, all'inattivazione rapida da parte di enzimi, alla scarsa selettività cellulare e spesso ad una scarsa solubilità in ambiente fisiologico, oltre a gravi effetti collaterali. Per cercare di ovviare, almeno in parte, a questi problemi, è stato preparato un coniugato polimerico direzionato al quale è stato legato un farmaco anti-cancro. Il coniugato migliora il profilo farmacocinetico del farmaco riducendo la clearance. Il “selective tumor targeting” può essere attivo o passivo. Il primo riguarda ligandi di recettori associati al tumore, che raggiungono il bersaglio sfruttando l’affinità ligando-recettore. Il secondo sistema può essere ottenuto sfruttando il cosiddetto effetto EPR (enhanced permeability and retention effect) grazie al quale molecole ad alto peso molecolare raggiungono e si accumulano nell’ambiente peritumorale. In questo lavoro è stato utilizzato un poli-(etilenglicole) eterobifunzionale legato ad epirubicina (EPI), un farmaco anti-cancro, e ad acido folico (FOL), come residuo di targeting. L'attività biologica del derivato FOL-PEG-EPI è stata studiata in due diversi sistemi di coltura, il classico sistema bi-dimensionale ed il sistema tri-dimensionale utilizzando Puramatrix hydrogelTM. Quest’ultimo dovrebbe ricreare un ambiente simile a quello in vivo. Gli studi di attività citotossica sono stati effettuati sulle seguenti linee cellulari: HT-29, MCF- 7 e KB-31 che presentano una diversa espressione del recettore di membrana per l’acido folico (rispettivamente normale espressione, medio-alta, alta). Lo studio di citotossicità su FOL-PEG-EPI ha mostrato maggiore tossicità su cellule KB-31, con sovra-espressione del recettore per l’acido folico, rispetto alle cellule MCF-7 e HT-29, sia in colture 2D che 3D. Inoltre, l’utilizzo del sistema di coltura tri-dimensionale ha dimostrato che FOL-PEG-EPI possiede attività selettiva sulle cellule KB-31, rispetto alle cellule HT-29 dove per ottenere l’IC50 è stata utilizzata una concentrazione di coniugato 3 volte più alta della massima utilizzabile in clinica. L’up-take cellulare dei coniugati ed epirubicina sono stati studiati mediante citofluorimetria e microscopia confocale. Nel primo caso, la citofluorimetria ha mostrato la presenza del segnale di fluorescenza all'interno delle cellule sia per FOL-PEG-EPI che per epirubicina. L'analisi di microscopia confocale ha confermato l’internalizzazione, localizzando in zona nucleare il farmaco libero ed in zona perinucleare il coniugato. Microsfere L’ingegneria dei tessuti è un campo interdisciplinare che applica i principi dell’ingegneria e delle scienze della vita allo sviluppo di sostituti biologici per ristabilire, mantenere o migliorare la funzione di tessuti e organi danneggiati. In questa ricerca si fondono discipline di biologia cellulare, ingegneria, scienza dei materiali e chirurgia allo scopo di costruire, mediante la combinazione di cellule, materiali (“scaffold”) e fattori di crescita, nuovi tessuti funzionali. I fattori di crescita possono essere impiegati per riprodurre le condizioni fisiologiche che consentono alle cellule di crescere, moltiplicarsi e differenziarsi nei diversi tipi di tessuti, ma la loro somministrazione rimane ancora una sfida tecnologica a causa della loro breve emivita nonché della loro difficoltà nel raggiungere il sito di targeting. La seconda parte di questo studio ha riguardato lo sviluppo di un sistema di Drug Delivery applicato all’ingegneria tissutale del tessuto osseo. La ricerca ha coinvolto l'utilizzo di un sistema di veicolazione di farmaci per il rilascio controllato della proteina TAT-OP1, che stimola la differenziazione osteogenica. Osteogenic protein-1 (OP-1 o BMP-7) è un membro della famiglia delle proteine morfogeniche dell’osso (bone morphogenic proteins, BMPs). Le BMP vengono riconosciute come fattori di crescita osteoinduttivi, ovvero promotori della formazione di nuovo tessuto osseo e appartengono alla superfamiglia del TGF-β. Le BMP sono secreti come precursori circa quattro volte più lunghi rispetto alla forma matura e possiedono una porzione C-terminale distintiva (pattern .. C ... CXGXC ... CC ... CXCX ..) contenente sette cisteine che costituiscono la regione attiva di queste proteine. In questo studio è stata utilizzata una proteina ricombinante di fusione chiamata TAT-OP1 che comprende una sequenza TAT, un peptide ricco di arginina derivante dall’HIV e che permette l’internalizzazione. Il costrutto TAT-OP1, di 162 aminoacidi, comprende: una porzione N-terminale 6 His-tag seguito dalla sequenza TAT, un sito di cleavage peptidasi-specifico (spanning 6 AA) e il C-terminale con il dominio OP-1 (126 AA) contenente il motivo di cisteine. Quando questo tipo di molecola bioattiva viene iniettato direttamente nel sito di azione, viene sottoposta ad inattivazione e rapida diluizione; questo ne limita l'uso in vivo. Per ovviare al problema sono state impiegate microsfere di poli-lattidecoglicolide (PLGA) per permettere un rilascio controllato di TAT-OP1 con l'obiettivo di mantenere un livello adeguato della proteina per tempi prolungati, migliorandone l’efficienza. Il rilascio delle molecole bioattive può essere facilmente modulato settando i parametri nella formulazione e nella tecnica di produzione. La tecnica dello spray drying è stata utilizzata per ottenere le microsfere con TAT-OP1. Il rilascio dalle microsfere con TAT-OP1 è stato studiato in un periodo di 7 giorni e l'efficienza di incapsulamento era risultata del 35%. Le dimensioni al microscopio a scansione elettronica (SEM) risultavano comprese tra 0,2-2 µm. Lo studio dell’attività biologica su microsfere con TAT- OP1, è stato condotto utilizzando pre-osteoblasti MC3T3-E1 a due diverse concentrazioni, 200 e 27 nM. Dopo 7 e 14 giorni di trattamento, le cellule mostravano presenza di mineralizzazione della matrice, test per la fosfatasi alcalina positivo e presenza di caratteristici marcatori osteogenici, quali osteopontina e osteocalcina. Questi risultati positivi ci hanno portato a valutare l'attività biologica della TAT- OP1 in microsfere in un sistema tri-dimensionale utilizzando cellule staminali mesenchimali isolate dal sangue del cordone ombelicale (UCBMSC). Il modello 3D è stata ottenuto utilizzando la matrice sintetica Puramatrix hydrogelTM, che è in grado di simulare il microambiente fisiologico. A seguito dell’incapsulazione di TAT-OP1 libera o di microsfere con TAT-OP1 in Puramatrix hydrogelTM, la risposta cellulare alla stimolazione di TAT-OP1 è stata valutata grazie all'analisi di microscopia elettronica a trasmissione (TEM) per rilevare la produzione di matrice ossea. Dopo 27 giorni di stimolazione con TAT-OP1 (200 nM), si osservava la presenza di microfibrille parzialmente aggregate attorno alle cellule. Depositi di calcio e cristalli di idrossiapatite sono stati rilevati solo in culture trattate con microsfere a rilascio controllato di TAT-OP1 (200nM). Pertanto, il rilascio controllato di TAT-OP1 da microsfere di PLGA sembra aumentare l’efficacia di stimolazione. Future indagini saranno dirette a confermare ulteriormente la capacità del presente approccio nel migliorare lo studio di differenziamento osteogenico in vitro e l'attività biologica della TAT-OP1 per un eventuale applicazione clinica nel campo dell’ingegneria tissutale dell’osso.