Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Edith Mathiowitz. Includes bibliographical references.

Bone grafting is an integral part of reconstructive surgery. In the United States alone over 250,000 bone grafts were harvested annually. While autogenic bone grafting has always been associated with donor site morbidity, bone graft substitutes have been suggested as a solution. In this project, a bone graft substitute using human dental pulp cells (HDPCs) and peptide nanofibre hydrogel was being developed. HDPCs were isolated from extracted teeth. After culture and expansion, unsorted HDPCs were encapsulated into peptide nanofibre hydrogel. These cell-gel constructs were cultured for two weeks in ordinary culture medium and then for 2-3 more weeks in osteogenic lineage induction medium. The post-induced cell-gel derived constructs were transplanted into skin pouches or calvarial bone defects of nude mice. When transplanted subcutaneously, the cell-gel derived constructs were harvested at four to twelve weeks postoperatively (n=5). Tissue samples were processed for contact radiograph, histological examination and antibody staining. These constructs developed into vascularised, mineralised tissue pieces. Though bone marker proteins (osteopontin, osteocalcin and osteonectin) were detected in these tissue pieces, the histological structure of their tissue matrix did not resemble bone matrix. Later, it was accidentally noted that portions of constructs touching the bone defect margin, would form bone through direct matrix transformation. This indicated that the current cell-gel model was potentially the first study model of tissue engineering bone by simulating intramembranous ossification. In the bone defect trial, obviously mineralized cell-gel derived constructs of matching shape and size were selected to patch the 3mm calvarial bone defects (n=5). Bone defect specimens were harvested at two weeks post-operation. The development of radio-opaque structures within the bone defects were evaluated in virtual 3-dimensional models constructed with data collected by microtomographic scanning. The bone nature of these radio-opaque structure were validated histologically (by staining with Hematoxylin and Eosin, Periodic acid-Schiff stain and Picrosirius red) and immunologically (with antibody against human collagen-I, osteonectin and parathyroid hormone receptor). The radio-opaque structures developed into the bone defect were evaluated positive for bone. And significantly more bone regeneration was observed in the test group (n=4) than in the control (n=2). The mean area percentages of regeneration were 46.3% and 0% respectively (p< 0.05). While the majority of studies in bone tissue engineering have worked with bone marrow stromal cells and scaffolds of synthetic polymer or calcium based materials, this is the first successful attempt of using HDPCs and peptide nanofibre hydrogel to engineer bone (in a nude mice mode). And the potential of these cell-gel derived constructs to promote bone regeneration was demonstrated. But this was the result from a single experiment of small sample size in one animal model only. It needs to be fortified by further experiments with larger population size and in other animal models. To increase clinical usefulness, the construct will need to be scaled up to centimetre size level. This will necessitate a change of its configuration from bead into meshwork. And, the data collected to date will shed light onto the redevelopment of all the relevant protocols. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Tissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures. / text

In previous in vitro and in vivo studies, decellularized adipose tissue (DAT) has demonstrated unique bioactivity, but little is known about the bioactive components preserved in the decellularized scaffold. With the goal of characterizing the bioactive components in the DAT, protein was extracted from DAT samples from 3 donors using 5 different buffers. The resulting DAT extracts were found to have very low protein content so molecular weight fractioning centrifugation was used to concentrate the samples. Concentrated extracts were screened for the presence of the bioactive components adiponectin, vascular endothelial growth factor A (VEGF-A), bone morphogenetic protein 2 (BMP-2) and Dickkopf related protein 1 (DKK-1) using Western blotting. Positive signal for BMP-2 was found for one donor in Roger’s Sample Buffer and Urea Buffer, but all other proteins investigated with Western blotting went undetected for all extraction buffers. Immunohistochemistry (IHC) was also used to determine the presence and distribution of bioactive components in the DAT samples. Sections of DAT were probed for the same bioactive components; adiponectin, VEGF-A, BMP-2 and DKK-1, with positive signals detected for adiponectin and VEGF-A. In order to develop an injectable, DAT-derived hydrogel for soft tissue regeneration, DATgels were fabricated from an enzymatically treated and homogenized DAT pre-gel suspension, which was neutralized to physiological pH and salt concentration. The highly-hydrated DATgels, containing up to 97% water, were found to degrade substantially over 14 days in simulated physiological fluid, with only a slight reduction in the overall scaffold size. Histology and SEM showed no major structural differences between the two formulations evaluated (40 mg/mL and 50 mg/mL), with both containing intact DAT features and small void spaces scattered heterogeneously throughout the scaffold. Preliminary in vitro cell work with adipose-derived stem cells (ASCs) showed that the 40 mg/mL formulation DATgel supported cell attachment and viability greater than 70% for all scaffolds up to 7 days after seeding. Further, migration into the scaffold was observed over time, indicating that the adhesive properties of the native ECM were retained through the processing steps required to fabricate the DATgels. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-12-19 16:22:32.717

Degenerative disc disease (DDD) is a common medical issue among human adults, leading to back pain and potentially, disability, decreasing an individual’s quality of life. In the United States alone, huge economic impacts are apparent with an estimated $50- 100 billion attributed to lost productivity and medical costs related to DDD. Spinal degeneration occurs in the intervertebral disc (IVD) and once damaged, the IVD is incapable of adequate self-repair. A regenerative therapy incorporating nucleus pulposus (NP) tissue engineering may provide an answer to spinal degeneration. The objective of this in vitro study was to evaluate the potential of a thiol-modified hyaluronan (TMHA) and elastin-like polypeptide (ELP) as a hydrogel scaffold for nucleus pulposus tissue engineering. Two materials, one composed of TMHA only and one a 3:1 TMHA/ELP, crosslinked with polyethylene glycol diacrylate (PEGDA), were seeded with cultured human NP cells and cyclic hydrostatic loading was applied at 1MPa for 3 hours a day for 3 consecutive days. Cell viability and gene expression were analyzed. A decreasing trend in cell viability with time and cyclic hydrostatic pressure loading was observed and statistically significant differences were observed between the TMHA unloaded treatment group at day 0 and the TMHA loaded treatment group at day 4 and between the TMHA unloaded treatment group at day 0 and the 3:1 TMHA/ELP loaded group at day 4. Comparisons between TMHA only and 3:1 TMHA/ELP hydrogels for the same treatment indicate similar trends and no statistically significant differences in biological effects were observed. Gene expression analysis indicated low frequency expression of NP extracellular matrix (ECM) molecules regardless of time point or cyclic hydrostatic pressure application. These results are revealing in that the 3:1 TMHA/ELP hydrogel did not support NP cells significantly better than the TMHA hydrogel, though cell source and hydrostatic pressure generation issues may have impacted this finding. Additional studies with alternative cell type and a refined hydrostatic pressure application method may better illuminate the efficacy of a 3:1 TMHA/ELP hydrogel as for NP tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-03-17 14:16:28.83

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

A composite bioscaffold was constructed by encapsulating human decellularized adipose tissue (DAT) within a photopolymerized polysaccharide hydrogel towards the goal of forming an injectable scaffold for adipose tissue engineering. Methacrylated glycol chitosan (MGC) and methacrylated chondroitin sulphate (MCS) were investigated as the hydrogel base materials with varying DAT concentrations. Glycol chitosan and chondroitin sulphate were converted to photopolymerizable prepolymers through graft methacrylation using glycidyl methacrylate and methacrylate anhydride respectively to achieve a degree of substitution (DOS) of 15% and 16%, respectively. MGC and MCS gels containing 0, 3 and 5 w/v% cryo-milled DAT were fabricated and characterized by measuring sol content, equilibrium water content and compressive mechanical properties (n=4, n=3). An increase in stiffness and a decrease in sol and water contents were observed in the gels with higher DAT concentration, suggesting that the DAT was acting as a filler material that contributed to the crosslinking reaction. In vitro studies were conducted with primary human adipose-derived stem cells (ASCs) encapsulated in the DAT-polymer constructs to assess cellular viability (n=3, N=3) as well as adipogenic differentiation, quantitatively via glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity (n=3, N=3) and qualitatively through end-point RT-PCR analysis of key adipogenic genes (LPL, PPARγ, and CEPBα) (n=2, N=3) and intracellular lipid staining (n=3, N=3). Incorporating the DAT with MGC or MCS hydrogels enhanced cell viability as compared to the MGC and MCS scaffolds alone, with the MCS + 5 w/v% DAT scaffold having the highest overall cell viability and total cell number. The addition of the DAT in the MGC and MCS scaffold groups enhanced ASC adipogenesis as measured by an increase in GPDH levels, adipogenic gene expression and intracellular lipid accumulation characteristic of adipocytes. The highest GPDH levels were observed in the induced MCS with 5 w/v% DAT scaffolds, as compared to all other scaffold groups and tissue culture controls. The GPDH activity in this group increased by almost three times between 3 and 14 days, consistent with the progression of differentiation. The results indicated that the MCS-based scaffolds incorporating the DAT promoted cell viability and adipogenesis, demonstrating great promise as composite scaffolds for soft tissue regeneration. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-12-23 15:12:37.772

Thesis (Ph.D.)--Tufts University, 2005. / Adviser: Gregory H. Altman. Submitted to the Dept. of Biology--Biotechnology. Includes bibliographical references (leaves 183-192). Access restricted to members of the Tufts University community. Also available via the World Wide Web;