Vascular-centric mapping of in vivo blood oxygen saturation in preclinical models.

Assessing intravascular blood oxygen saturation (SO2) is crucial for characterizing in vivo microenvironmental changes in preclinical models of injury and disease. However, most conventional optical imaging techniques for mapping in vivo SO2 assume or compute a single value of the optical path-length in tissue. This is especially detrimental when mapping in vivo SO2 in experimental disease or wound healing models that are characterized by vascular and tissue remodeling. Therefore, to circumvent this limitation we developed an in vivo SO2 mapping technique that utilizes hemoglobin-based intrinsic optical signal (IOS) imaging combined with a vascular-centric estimation of optical path-lengths. In vivo arterial and venous SO2 distributions derived with this approach closely matched those reported in the literature, while those derived using the single path-length (i.e. conventional) approach did not. Moreover, in vivo cerebrovascular SO2 strongly correlated (R2 > 0.7) with changes in systemic SO2 measured with a pulse oximeter during hypoxia and hyperoxia paradigms. Finally, in a calvarial bone healing model, in vivo SO2 assessed over four weeks was spatiotemporally correlated with angiogenesis and osteogenesis (R2 > 0.6). During the early stages of bone healing (i.e. day 10), angiogenic vessels surrounding the calvarial defect exhibited mean SO2 that was elevated by10 % (p < 0.05) relative to that observed at a later stage (i.e., day 26), indicative of their role in osteogenesis. These correlations were not evident with the conventional SO2 mapping approach. The feasibility of our wide field-of-view in vivo SO2 mapping approach illustrates its potential for characterizing the microvascular environment in applications ranging from tissue engineering to cancer.

Quantitative 3D imaging of the cranial microvascular environment at single-cell resolution.

Vascularization is critical for skull development, maintenance, and healing. Yet, there remains a significant knowledge gap in the relationship of blood vessels to cranial skeletal progenitors during these processes. Here, we introduce a quantitative 3D imaging platform to enable the visualization and analysis of high-resolution data sets (>100 GB) throughout the entire murine calvarium. Using this technique, we provide single-cell resolution 3D maps of vessel phenotypes and skeletal progenitors in the frontoparietal cranial bones. Through these high-resolution data sets, we demonstrate that CD31hiEmcnhi vessels are spatially correlated with both Osterix+ and Gli1+ skeletal progenitors during postnatal growth, healing, and stimulated remodeling, and are concentrated at transcortical canals and osteogenic fronts. Interestingly, we find that this relationship is weakened in mice with a conditional knockout of PDGF-BB in TRAP+ osteoclasts, suggesting a potential role for osteoclasts in maintaining the native cranial microvascular environment. Our findings provide a foundational framework for understanding how blood vessels and skeletal progenitors spatially interact in cranial bone, and will enable more targeted studies into the mechanisms of skull disease pathologies and treatments. Additionally, our technique can be readily adapted to study numerous cell types and investigate other elusive phenomena in cranial bone biology.

Effects of Single-Dose Versus Hypofractionated Focused Radiation on Vertebral Body Structure and Biomechanical Integrity: Development of a Rabbit Radiation-Induced Vertebral Compression Fracture Model.

PURPOSE: Vertebral compression fracture is a common complication of spinal stereotactic body radiation therapy. Development of an in vivo model is crucial to fully understand how focal radiation treatment affects vertebral integrity and biology at various dose fractionation regimens. We present a clinically relevant animal model to analyze the effects of localized, high-dose radiation on vertebral microstructure and mechanical integrity. Using this model, we test the hypothesis that fractionation of radiation dosing can reduce focused radiation therapy's harmful effects on the spine. METHODS AND MATERIALS: The L5 vertebra of New Zealand white rabbits was treated with either a 24-Gy single dose of focused radiation or 3 fractionated 8-Gy doses over 3 consecutive days via the Small Animal Radiation Research Platform. Nonirradiated rabbits were used as controls. Rabbits were euthanized 6 months after irradiation, and their lumbar vertebrae were harvested for radiologic, histologic, and biomechanical testing. RESULTS: Localized single-dose radiation led to decreased vertebral bone volume and trabecular number and a subsequent increase in trabecular spacing and thickness at L5. Hypofractionation of the radiation dose similarly led to reduced trabecular number and increased trabecular spacing and thickness, yet it preserved normalized bone volume. Single-dose irradiated vertebrae displayed lower fracture loads and stiffness compared with those receiving hypofractionated irradiation and with controls. The hypofractionated and control groups exhibited similar fracture load and stiffness. For all vertebral samples, bone volume, trabecular number, and trabecular spacing were correlated with fracture loads and Young's modulus (P < .05). Hypocellularity was observed in the bone marrow of both irradiated groups, but osteogenic features were conserved in only the hypofractionated group. CONCLUSIONS: Single-dose focal irradiation showed greater detrimental effects than hypofractionation on the microarchitectural, cellular, and biomechanical characteristics of irradiated vertebral bodies. Correlation between radiologic measurements and biomechanical properties supported the reliability of this animal model of radiation-induced vertebral compression fracture, a finding that can be applied to future studies of preventative measures.

Comparison of Freshly Isolated Adipose Tissue-derived Stromal Vascular Fraction and Bone Marrow Cells in a Posterolateral Lumbar Spinal Fusion Model.

STUDY DESIGN: Rat posterolateral lumbar fusion model. OBJECTIVE: The aim of this study was to compare the efficacy of freshly isolated adipose tissue-derived stromal vascular fraction (A-SVF) and bone marrow cells (BMCs) cells in achieving spinal fusion in a rat model. SUMMARY OF BACKGROUND DATA: Adipose tissue-derived stromal cells (ASCs) offer advantages as a clinical cell source compared to bone marrow-derived stromal cells (BMSCs), including larger available tissue volumes and reduced donor site morbidity. While pre-clinical studies have shown that ex vivo expanded ASCs can be successfully used in spinal fusion, the use of A-SVF cells better allows for clinical translation. METHODS: A-SVF cells were isolated from the inguinal fat pads, whereas BMCs were isolated from the long bones of syngeneic 6- to 8-week-old Lewis rats and combined with Vitoss (Stryker) bone graft substitute for subsequent transplantation. Posterolateral spinal fusion surgery at L4-L5 was performed on 36 female Lewis rats divided into three experimental groups: Vitoss bone graft substitute only (VO group); Vitoss + 2.5 × 106 A-SVF cells/side; and, Vitoss + 2.5 × 106 BMCs/side. Fusion was assessed 8 weeks post-surgery via manual palpation, micro-computed tomography (µCT) imaging, and histology. RESULTS: µCT imaging analyses revealed that fusion volumes and µCT fusion scores in the A-SVF group were significantly higher than in the VO group; however, they were not significantly different between the A-SVF group and the BMC group. The average manual palpation score was highest in the A-SVF group compared with the BMC and VO groups. Fusion masses arising from cell-seeded implants yielded better bone quality than nonseeded bone graft substitute. CONCLUSION: In a rat model, A-SVF cells yielded a comparable fusion mass volume and radiographic rate of fusion to BMCs when combined with a clinical-grade bone graft substitute. These results suggest the feasibility of using freshly isolated A-SVF cells in spinal fusion procedures.Level of Evidence: N/A.

Non-viral gene delivery of HIF-1α promotes angiogenesis in human adipose-derived stem cells.

Stable and mature vascular formation is a current challenge in engineering functional tissues. Transient, non-viral gene delivery presents a unique platform for delivering genetic information to cells for tissue engineering purposes and to restore blood flow to ischemic tissue. The formation of new blood vessels can be induced by upregulation of hypoxia-inducible factor-1α (HIF-1), among other factors. We hypothesized that biodegradable polymers could be used to efficiently deliver the HIF-1α gene to human adipose-derived stromal/stem cells (hASCs) and that this treatment could recruit an existing endogenous endothelial cell population to induce angiogenesis in a 3D cell construct in vitro. In this study, end-modified poly(ß-amino ester) (PBAE) nanocomplexes were first optimized for transfection of hASCs and a new biodegradable polymer with increased hydrophobicity and secondary amine structures, N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine end-modified poly(1,4-butanediol diacrylate-co-4-amino-1-butanol), was found to be most effective. Optimal PBAE nanocomplexes had a hydrodynamic diameter of approximately 140 nm and had a zeta potential of 30 mV. The PBAE polymer self-assembled with HIF-1α plasmid DNA and treatment of hASCs with these nanocomplexes induced 3D vascularization. Cells transfected with this polymer-DNA complex were found to have 106-fold upregulation HIF-1α expression, an approximately 2-fold increase in secreted VEGF, and caused the formation of vessel tubules compared to an untransfected control. These gene therapy biomaterials may be useful for regenerative medicine. STATEMENT OF SIGNIFICANCE: Not only is the formation of stable vasculature a challenge for engineering human tissues in vitro, but it is also of valuable interest to clinical applications such as peripheral artery disease. Previous studies using HIF-1α to induce vascular formation have been limited by the necessity of hypoxic chambers. It would be advantageous to simulate endogenous responses to hypoxia without the need for physical hypoxia. In this study, 3D vascular formation was shown to be inducible through non-viral gene delivery of HIF-1α with new polymeric nanocomplexes. A biodegradable polymer N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine end-modified poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) demonstrates improved transfection of human adipose-derived stem cells. This nanobiotechnology could be a promising strategy for the creation of vasculature for tissue engineering and clinical applications.

Nanofiber-hydrogel composite-mediated angiogenesis for soft tissue reconstruction.

Soft tissue losses from tumor removal, trauma, aging, and congenital malformation affect millions of people each year. Existing options for soft tissue restoration have several drawbacks: Surgical options such as the use of autologous tissue flaps lead to donor site defects, prosthetic implants are prone to foreign body response leading to fibrosis, and fat grafting and dermal fillers are limited to small-volume defects and only provide transient volume restoration. In addition, large-volume fat grafting and other tissue-engineering attempts are hampered by poor vascular ingrowth. Currently, there are no off-the-shelf materials that can fill the volume lost in soft tissue defects while promoting early angiogenesis. Here, we report a nanofiber-hydrogel composite that addresses these issues. By incorporating interfacial bonding between electrospun poly(ε-caprolactone) fibers and a hyaluronic acid hydrogel network, we generated a composite that mimics the microarchitecture and mechanical properties of soft tissue extracellular matrix. Upon subcutaneous injection in a rat model, this composite permitted infiltration of host macrophages and conditioned them into the pro-regenerative phenotype. By secreting pro-angiogenic cytokines and growth factors, these polarized macrophages enabled gradual remodeling and replacement of the composite with vascularized soft tissue. Such host cell infiltration and angiogenesis were also observed in a rabbit model for repairing a soft tissue defect filled with the composite. This injectable nanofiber-hydrogel composite augments native tissue regenerative responses, thus enabling durable soft tissue restoration outcomes.

Heparin-Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF-BB to Adipose-Derived Stem Cells in Tissue Engineered Bone Grafts.

Adipose-derived stem cells (ASCs) are a promising cell source for regenerating critical-sized craniofacial bone defects, but their clinical use is limited due to the supraphysiological levels of bone morphogenetic protein-2 required to induce bone formation in large grafts. It has been recently reported that platelet-derived growth factor-BB (PDGF) directly enhances the osteogenesis of ASCs when applied at physiological concentrations. In this study, a biomimetic delivery system that tethers PDGF to decellularized bone matrix (DCB) is developed to enhance osteogenic signaling in bone grafts by colocalizing PDGF-extracellular matrix cues. Heparin is conjugated to DCB particles (HC-DCB) to promote sustained binding of PDGF via electrostatic interactions. HC-DCB particles bind to PDGF with >99% efficiency and release significantly less PDGF over 21 days compared to nonconjugated DCB particles (1.1% vs 22.8%). HC-DCB-PDGF signaling in polycaprolactone (PCL)-fibrin grafts promotes >40 µg Ca2+ µg-1 DNA deposition by ASCs during in vitro osteogenic culture compared to grafts without HC-DCB or PDGF. Furthermore, more bone formation is observed in grafts with HC-DCB-PDGF at 12 weeks following implantation of grafts into murine critical-sized calvarial defects. Collectively, these results demonstrate that HC-DCB enhances the osteogenic signaling of PDGF to ASCs and may be applied to promote ASC-mediated bone regeneration in critical-sized defects.

Adipose-derived Stem/Stromal Cells on Electrospun Fibrin Microfiber Bundles Enable Moderate Muscle Reconstruction in a Volumetric Muscle Loss Model.

Current treatment options for volumetric muscle loss (VML) are limited due to donor site morbidity, lack of donor tissue, and insufficient functional recovery. Tissue-engineered skeletal muscle grafts offer the potential to significantly improve functional outcomes. In this study, we assessed the potential pro-myogenic effects of human adipose-derived stem cells (ASCs) seeded onto electrospun uniaxially aligned fibrin hydrogel microfiber bundles. Although both uninduced and 5-azacytidine-induced ASCs exhibited alignment, elongation, and diffuse muscle marker expression when grown on microfiber bundles for 2 months in vitro, both groups failed to fully recapitulate myotube characteristics. To assess the muscle regeneration potential of ASCs in vivo, ASC-seeded fibrin microfiber bundles were implanted in a robust murine VML defect model. Minimal fibrosis was observed surrounding implanted acellular hydrogel fibers at 2 and 4 weeks, and fibers seeded with ASCs exhibited up to 4 times higher volume retention than acellular fibers. We observed increased numbers of cells positive for the regenerating muscle marker embryonic myosin and the mature muscle marker myosin heavy chain in ASC-seeded fibers compared with acellular fibers at 1 and 3 months post-transplantation. Regenerating muscle cells were closely associated with ASC-derived cells and in some cases had potentially fused with them. These findings demonstrate that despite failing to undergo myogenesis in vitro, ASCs combined with electrospun fibrin microfibers moderately increased muscle reconstruction in vivo compared with acellular fibers following a severe VML defect.

Advances in Perfusion Systems for Solid Organ Preservation.

In the past, a diagnosis of organ failure would essentially be a death sentence for patients. With improved techniques for organ procurement and surgical procedures, transplantations to treat organ failure have become standard medical practice. However, while the demand for organs has skyrocketed, the donor pool has not kept pace leading to long recipient waiting lists. Organ preservation provides a means to increase the number of available transplantable organs. However, there are significant drawbacks associated with cold storage, the current gold standard. To address the short-comings due to diffusional limitations, engineers have developed cold perfusion systems. More recently, there has been a significant trend towards the development of near-normothermic systems to enhance the functional preservation of solid organs including livers, lungs, hearts, kidneys, and vascularized composite allotransplants. Here we review recent advances in the development of perfusion systems for the preservation of solid organs. We provide a brief history of organ transplantation, the limitations of existing systems, and describe research being done to develop commercially available perfusion systems to enhance organ preservation.

Phenotyping the Microvasculature in Critical-Sized Calvarial Defects via Multimodal Optical Imaging.

Tissue-engineered scaffolds are a powerful means of healing craniofacial bone defects arising from trauma or disease. Murine models of critical-sized bone defects are especially useful in understanding the role of microenvironmental factors such as vascularization on bone regeneration. Here, we demonstrate the capability of a novel multimodality imaging platform capable of acquiring in vivo images of microvascular architecture, microvascular blood flow, and tracer/cell tracking via intrinsic optical signaling (IOS), laser speckle contrast (LSC), and fluorescence (FL) imaging, respectively, in a critical-sized calvarial defect model. Defects that were 4 mm in diameter were made in the calvarial regions of mice followed by the implantation of osteoconductive scaffolds loaded with human adipose-derived stem cells embedded in fibrin gel. Using IOS imaging, we were able to visualize microvascular angiogenesis at the graft site and extracted morphological information such as vessel radius, length, and tortuosity two weeks after scaffold implantation. FL imaging allowed us to assess functional characteristics of the angiogenic vessel bed, such as time-to-peak of a fluorescent tracer, and also allowed us to track the distribution of fluorescently tagged human umbilical vein endothelial cells. Finally, we used LSC to characterize the in vivo hemodynamic response and maturity of the remodeled microvessels in the scaffold microenvironment. In this study, we provide a methodical framework for imaging tissue-engineered scaffolds, processing the images to extract key microenvironmental parameters, and visualizing these data in a manner that enables the characterization of the vascular phenotype and its effect on bone regeneration. Such multimodality imaging platforms can inform optimization and design of tissue-engineered scaffolds and elucidate the factors that promote enhanced vascularization and bone formation.

Myogenic Differentiation of ASCs Using Biochemical and Biophysical Induction.

Adipose-derived stem/stromal cells (ASCs) constitute a very promising source for cell therapy and tissue engineering approaches as they can be easily obtained in large quantities with comparatively minimal patient discomfort. Moreover, ASCs have multilineage differentiation capacity. Among these, differentiation capacity along the myogenic lineage is of particular interest since myogenic precursors are scarce and obtaining a large number of cells from skeletal muscle biopsies is difficult. Here, we describe a method to effectively induce ASC myogenesis through the combination of biochemical (cocktail including 5-azacytidine and horse serum) and biophysical (dynamic culture via uniaxial cyclic strain) stimulation. This method results in multinucleated cells that are positive in myogenic markers including Pax 3/7, desmin, myoD, and myosin heavy chain.

3D-Printing Composite Polycaprolactone-Decellularized Bone Matrix Scaffolds for Bone Tissue Engineering Applications.

Millions of patients worldwide require bone grafts for treatment of large, critically sized bone defects from conditions such as trauma, cancer, and congenital defects. Tissue engineered (TE) bone grafts have the potential to provide a more effective treatment than current bone grafts since they would restore fully functional bone tissue in large defects. Most bone TE approaches involve a combination of stem cells with porous, biodegradable scaffolds that provide mechanical support and degrade gradually as bone tissue is regenerated by stem cells. 3D-printing is a key technique in bone TE that can be used to fabricate functionalized scaffolds with patient-specific geometry. Using 3D-printing, composite polycaprolactone (PCL) and decellularized bone matrix (DCB) scaffolds can be produced to have the desired mechanical properties, geometry, and osteoinductivity needed for a TE bone graft. This book chapter will describe the protocols for fabricating and characterizing 3D-printed PCL:DCB scaffolds. Moreover, procedures for culturing adipose-derived stem cells (ASCs) in these scaffolds in vitro will be described to demonstrate the osteoinductivity of the scaffolds.

Adipose-derived perivascular mesenchymal stromal/stem cells promote functional vascular tissue engineering for cardiac regenerative purposes.

Cardiac tissue engineering approaches have the potential to regenerate functional myocardium with intrinsic vascular networks. This study compared the relative effects of human adipose-derived stem/stromal cells (hASCs) and human dermal fibroblasts (hDFs) in cocultures with neonatal rat ventricular cardiomyocytes (NRVCMs) and human umbilical vein endothelial cells (HUVECs). At the same ratios of NRVCM:hASC and NRVCM:hDF, the hASC cocultures displayed shorter action potentials and maintained capture at faster pacing rates. Similarly, in coculture with HUVECs, hASC:HUVEC exhibited superior ability to support vascular capillary network formation relative to hDF:HUVEC. Based on these studies, a range of suitable cell ratios were determined to develop a triculture system. Six seeding ratios of NRVCM:hASC:HUVEC were tested and it was found that a ratio of 500:50:25 cells (i.e. 250,000:25,000:12,500 cells/cm2 ) resulted in the formation of robust vascular networks while retaining action potential durations and propagation similar to pure NRVCM cultures. Tricultures in this ratio exhibited an average conduction velocity of 20 ± 2 cm/s, action potential durations at 80% repolarization (APD80 ) and APD30 of 122 ± 5 ms and 59 ± 4 ms, respectively, and maximum capture rate of 7.4 ± 0.6 Hz. The NRVCM control groups had APD80 and APD30 of 120 ± 9 ms and 51 ± 5 ms, with a maximum capture rate of 7.3 ± 0.2 Hz. In summary, the combination of hASCs in the appropriate ratios with NRVCMs and HUVECs can facilitate the formation of densely vascularized cardiac tissues that appear not to impact the electrophysiological function of cardiomyocytes negatively. Copyright © 2017 John Wiley & Sons, Ltd.

Recent Advances in Tissue Engineering Strategies for the Treatment of Joint Damage.

PURPOSE OF REVIEW: While the clinical potential of tissue engineering for treating joint damage has yet to be realized, research and commercialization efforts in the field are geared towards overcoming major obstacles to clinical translation, as well as towards achieving engineered grafts that recapitulate the unique structures, function, and physiology of the joint. In this review, we describe recent advances in technologies aimed at obtaining biomaterials, stem cells, and bioreactors that will enable the development of effective tissue-engineered treatments for repairing joint damage. RECENT FINDINGS: 3D printing of scaffolds is aimed at improving the mechanical structure and microenvironment necessary for bone regeneration within a damaged joint. Advances in our understanding of stem cell biology and cell manufacturing processes are informing translational strategies for the therapeutic use of allogeneic and autologous cells. Finally, bioreactors used in combination with cells and biomaterials are promising strategies for generating large tissue grafts for repairing damaged tissues in pre-clinical models. Together, these advances along with ongoing research directions are making tissue engineering increasingly viable for the treatment of joint damage.

Stem Cell Fate Decision Making: Modeling Approaches.

Mathematical (computational) modeling approaches can be effective tools in providing insight into cell-fate decisions. In this article, several major approaches to the modeling of embryonic, hematopoietic, adipose-derived, cancer, and neural stem cell differentiation are discussed. First, the population dynamics approach is considered. The models described as bifurcating dynamical systems that result in bistability or periodic oscillations are then discussed. Also, spatiotemporal models of cell differentiation, including continuum and discrete (agent- and rule-based) approaches, are reviewed. Further, the effects of the mechanical factors are discussed, including the convergence of the differentiation and mechanotransducton pathways and computational analysis of the extracellular matrix (surrounding tissue). Finally, the stochastic models that take into account the molecular noise of internal and external origins are reviewed. The effectiveness of the modeling in the creation of the improved differentiation platforms, elucidation of various pathological conditions, and analysis of treatment regiments has been demonstrated.

3D-Printing Technologies for Craniofacial Rehabilitation, Reconstruction, and Regeneration.

The treatment of craniofacial defects can present many challenges due to the variety of tissue-specific requirements and the complexity of anatomical structures in that region. 3D-printing technologies provide clinicians, engineers and scientists with the ability to create patient-specific solutions for craniofacial defects. Currently, there are three key strategies that utilize these technologies to restore both appearance and function to patients: rehabilitation, reconstruction and regeneration. In rehabilitation, 3D-printing can be used to create prostheses to replace or cover damaged tissues. Reconstruction, through plastic surgery, can also leverage 3D-printing technologies to create custom cutting guides, fixation devices, practice models and implanted medical devices to improve patient outcomes. Regeneration of tissue attempts to replace defects with biological materials. 3D-printing can be used to create either scaffolds or living, cellular constructs to signal tissue-forming cells to regenerate defect regions. By integrating these three approaches, 3D-printing technologies afford the opportunity to develop personalized treatment plans and design-driven manufacturing solutions to improve aesthetic and functional outcomes for patients with craniofacial defects.

Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration.

Tissue-engineered approaches to regenerate bone in the craniomaxillofacial region utilize biomaterial scaffolds to provide structural and biological cues to stem cells to stimulate osteogenic differentiation. Bioactive scaffolds are typically comprised of natural components but often lack the manufacturability of synthetic materials. To circumvent this trade-off, we 3D printed materials comprised of decellularized bone (DCB) matrix particles combined with polycaprolactone (PCL) to create novel hybrid DCB:PCL scaffolds for bone regeneration. Hybrid scaffolds were readily printable at compositions of up to 70% bone by mass and displayed robust mechanical properties. Assessments of surface features revealed both collagenous and mineral components of bone were present. Qualitative and quantitative assessments showed increased surface roughness relative to that of pure PCL scaffolds. These findings correlated with enhanced cell adhesion on hybrid surfaces relative to that on pure surfaces. Human adipose-derived stem cells (hASCs) cultured in DCB:PCL scaffolds without soluble osteogenic cues exhibited significant upregulation of osteogenic genes in hybrid scaffolds relative to pure PCL scaffolds. In the presence of soluble phosphate, hybrid scaffolds resulted in increased calcification. The hASC-seeded scaffolds were implanted into critical-sized murine calvarial defects and yielded greater bone regeneration in DCB:PCL scaffolds compared to that in PCL-only at 1 and 3 months post-transplantation. Taken together, these results demonstrate that 3D printed DCB:PCL scaffolds might be effective for stimulating bone regeneration.

Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRß within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

Oxygen delivery from hyperbarically loaded microtanks extends cell viability in anoxic environments.

Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo.

Stromal cells and stem cells in clinical bone regeneration.

Stem-cell-mediated bone repair has been used in clinical trials for the regeneration of large craniomaxillofacial defects, to slow the process of bone degeneration in patients with osteonecrosis of the femoral head and for prophylactic treatment of distal tibial fractures. Successful regenerative outcomes in these investigations have provided a solid foundation for wider use of stromal cells in skeletal repair therapy. However, employing stromal cells to facilitate or enhance bone repair is far from being adopted into clinical practice. Scientific, technical, practical and regulatory obstacles prevent the widespread therapeutic use of stromal cells. Ironically, one of the major challenges lies in the limited understanding of the mechanisms via which transplanted cells mediate regeneration. Animal models have been used to provide insight, but these models largely fail to reproduce the nuances of human diseases and bone defects. Consequently, the development of targeted approaches to optimize cell-mediated outcomes is difficult. In this Review, we highlight the successes and challenges reported in several clinical trials that involved the use of bone-marrow-derived mesenchymal or adipose-tissue-derived stromal cells. We identify several obstacles blocking the mainstream use of stromal cells to enhance skeletal repair and highlight technological innovations or areas in which novel techniques might be particularly fruitful in continuing to advance the field of skeletal regenerative medicine.