Sarita R. Shah

Poly(lactic acid) nanofibrous scaffolds for tissue engineering

Poly(lactic acid) (PLA) is a synthetic polyester that has shown extensive utility in tissue engineering. Synthesized either by ring opening polymerization or polycondensation, PLA hydrolytically degrades into lactic acid, a metabolic byproduct, making it suitable for medical applications. Specifically, PLA nanofibers have widened the possible uses of PLA scaffolds for regenerative medicine and drug delivery applications. The use of nanofibrous scaffolds imparts a host of desirable properties, including high surface area, biomimicry of native extracellular matrix architecture, and tuning of mechanical properties, all of which are important facets of designing scaffolds for a particular organ system. Additionally, nanofibrous PLA scaffolds hold great promise as drug delivery carriers, where fabrication parameters and drug-PLA compatibility greatly affect the drug release kinetics. In this review, we present the latest advances in the use of PLA nanofibrous scaffolds for musculoskeletal, nervous, cardiovascular, and cutaneous tissue engineering and offer perspectives on their future use.

Injectable dual-gelling cell-laden composite hydrogels for bone tissue engineering

The present work investigated the osteogenic potential of injectable, dual thermally and chemically gelable composite hydrogels for mesenchymal stem cell (MSC) delivery in vitro and in vivo. Composite hydrogels comprising copolymer macromers of N-isopropylacrylamide were fabricated through the incorporation of gelatin microparticles (GMPs) as enzymatically digestible porogens and sites for cellular attachment. High and low polymer content hydrogels with and without GMP loading were shown to successfully encapsulate viable MSCs and maintain their survival over 28 days in vitro. GMP incorporation was also shown to modulate alkaline phosphatase production, but enhanced hydrogel mineralization along with higher polymer content even in the absence of cells. Moreover, the regenerative capacity of 2 mm thick hydrogels with GMPs only, MSCs only, or GMPs and MSCs was evaluated in vivo in an 8 mm rat critical size cranial defect for 4 and 12 weeks. GMP incorporation led to enhanced bony bridging and mineralization within the defect at each timepoint, and direct bone-implant contact as determined by microcomputed tomography and histological scoring, respectively. Encapsulation of both GMPs and MSCs enabled hydrogel degradation leading to significant tissue infiltration and osteoid formation. The results suggest that these injectable, dual-gelling cell-laden composite hydrogels can facilitate bone ingrowth and integration, warranting further investigation for bone tissue engineering.

Osteochondral Tissue Regeneration Through Polymeric Delivery of DNA Encoding for the SOX Trio and RUNX2

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)-hyaluronic acid (bPEI-HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI-HA complexes. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI-HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.

Immunomodulatory properties of stem cells and bioactive molecules for tissue engineering.

The immune system plays a crucial role in the success of tissue engineering strategies. Failure to consider the interactions between implantable scaffolds, usually containing cells and/or bioactive molecules, and the immune system can result in rejection of the implant and devastating clinical consequences. However, recent research into mesenchymal stem cells, which are commonly used in many tissue engineering applications, indicates that they may play a beneficial role modulating the immune system. Likewise, direct delivery of bioactive molecules involved in the inflammatory process can promote the success of tissue engineering constructs. In this article, we will review the various mechanisms in which modulation of the immune system is achieved through delivered bioactive molecules and cells and contextualize this information for future strategies in tissue engineering.

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Incorporation of fast dissolving glucose porogens into an injectable calcium phosphate cement for bone tissue engineering

Calcium phosphate cements (CPCs) have been extensively investigated as scaffolds in bone tissue engineering in light of their chemical composition closely resembling the mineral component of bone extracellular matrix. Yet, the degradation kinetics of many CPCs is slow compared to de novo bone formation. In order to overcome this shortcoming, the use of porogens within CPCs has been suggested as a potential strategy to increase scaffold porosity and promote surface degradation. This study explored the usage of glucose microparticles (GMPs) as porogens for the introduction of macroporosity within CPCs, and characterized the handling properties and physicochemical characteristics of CPCs containing GMPs. Samples were fabricated with four different weight fractions of GMPs (10, 20, 30, and 40%) and two different size ranges (100-150μm and 150-300μm), and were assayed for porosity, pore size distribution, morphology, and compressive mechanical properties. Samples were further tested for their handling properties - specifically, setting time and cohesiveness. Additionally, these same analyses were conducted on samples exposed to a physiological solution in order to estimate the dissolution kinetics of GMPs and its effect on the properties of the composite. GMPs were efficiently encapsulated and homogeneously dispersed in the resulting composite. Although setting times increased for GMP/CPC formulations compared to control CPC material, increasing the Na2HPO4 concentration in the liquid phase decreased the initial setting time to clinically acceptable values (i.e. <15min). Incorporation of GMPs led to the formation of instant macroporosity upon cement setting, and encapsulated GMPs completely dissolved in three days, resulting in a further increase in scaffold porosity. However, the dissolution of GMPs decreased scaffold compressive strength. Overall, the introduction of GMPs into CPC resulted in macroporous scaffolds with good handling properties, as well as designer porosity and pore size distribution via selection of the appropriate size/weight fraction of GMPs. The data demonstrate that GMPs are promising porogens for the production of highly tunable porous CPC scaffolds. STATEMENT OF SIGNIFICANCE Calcium phosphate cements have shown great promise for the regeneration of bone. However, macropores (>100μm) are required for promoting bone ingrowth. Several studies have investigated methods to generate macroporosity within calcium phosphate cements but many of these methods either affect the cement setting or take weeks or months to generate the maximum porosity. This work offers a new method for generating macroporosity within calcium phosphate cements by utilizing glucose microparticles. The microparticles dissolve in less then 72h, thereby generating scaffolds with maximum porosity in short period of time. The results will offer a new method for generating macroporosity within calcium phosphate cements.

A composite critical-size rabbit mandibular defect for evaluation of craniofacial tissue regeneration

Translational biomaterials targeted toward the regeneration of large bone defects in the mandible require a preclinical model that accurately recapitulates the regenerative challenges present in humans. Computational modeling and in vitro assays do not fully replicate the in vivo environment. Consequently, in vivo models can have specific applications such as those of the mandibular angle defect, which is used to investigate bone regeneration in a nonload-bearing area, and the inferior border mandibular defect, which is a model for composite bone and nerve regeneration, with both models avoiding involvement of soft tissue or teeth. In this protocol, we describe a reproducible load-bearing critical-size composite tissue defect comprising loss of soft tissue, bone and tooth in the mandible of a rabbit. We have previously used this procedure to investigate bone regeneration, vascularization and infection prevention in response to new biomaterial formulations for craniofacial tissue engineering applications. This surgical approach can be adapted to investigate models such as that of regeneration in the context of osteoporosis or irradiation. The procedure can be performed by researchers with basic surgical skills such as dissection and suturing. The procedure takes 1.5–2 h, with ∼2 h of immediate postoperative care, and animals should be monitored daily for the remainder of the study. For bone tissue engineering applications, tissue collection typically occurs 12 weeks after surgery. In this protocol, we will present the necessary steps to ensure reproducibility; tips to minimize complications during and after surgery; and analytical techniques for assessing soft tissue, bone and vessel regeneration by gross evaluation, microcomputed tomography (microCT) and histology.

Infected Animal Models for Tissue Engineering

Infection is one of the most common complications associated with medical interventions and implants. As tissue engineering strategies to replace missing or damaged tissue advance, the focus on prevention and treatment of concomitant infection has also begun to emerge as an important area of research. Because the in vivo environment is a complex interaction between host tissue, implanted materials, and native immune system that cannot be replicated in vitro, animal models of infection are integral in evaluating the safety and efficacy of experimental treatments for infection. In this review, considerations for selecting an animal model, established models of infection, and areas that require further model development are discussed with regard to cutaneous, fascial, and orthopedic infections.

Use of Porous Space Maintainers in Staged Mandibular Reconstruction

The success of mandibular reconstructions depends not only on restoring the form and function of lost bone but also on the preservation of the overlying soft tissue layer. In this case study, 5 porous polymethylmethacrylate space maintainers fabricated via patient-specific molds were implanted initially to maintain the vitality of the overlying oral mucosa during staged mandibular reconstructions. Three of the 5 patients healed well; the other 2 patients developed dehiscences, likely due to a thin layer of soft tissue overlying the implant. The results presented provide evidence that a larger investigation of space maintainers fabricated using this method is warranted.

Reconstruction of large mandibular defects using autologous tissues generated from in vivo bioreactors

Reconstruction of large mandibular defects is clinically challenging due to the need for donor tissue of appropriate shape and volume to facilitate high fidelity repair. In order to generate large vascularized tissues of custom geometry, bioreactors were implanted against the rib periosteum of 3-4year-old sheep for nine weeks. Bioreactors were filled with either morcellized autologous bone, synthetic ceramic particles, or a combination thereof. Tissues generated within synthetic graft-filled bioreactors were transferred into a large right-sided mandibular angle defect as either avascular grafts (n=3) or vascularized free flaps (n=3). After twelve additional weeks, reconstructed mandibular angles were harvested and compared to contralateral control angles. Per histologic and radiologic evaluation, a greater amount of mineralized tissue was generated in bioreactors filled with autologous graft although the quality of viable bone was not significantly different between groups. Genetic analyses of soft tissue surrounding bioreactor-generated tissues demonstrated similar early and late stage osteogenic biomarker expression (Runx2 and Osteocalcin) between the bioreactors and rib periosteum. Although no significant differences between the height of reconstructed and control mandibular angles were observed, the reconstructed mandibles had decreased bone volume. There were no differences between mandibles reconstructed with bioreactor-generated tissues transferred as flaps or grafts. Tissues used for mandibular reconstruction demonstrated integration with native bone as well as evidence of remodeling. In this study, we have demonstrated that synthetic scaffolds are sufficient to generate large volumes of mineralized tissue in an in vivo bioreactor for mandibular reconstruction. STATEMENT OF SIGNIFICANCE A significant clinical challenge in craniofacial surgery is the reconstruction of large mandibular defects. In this work, we demonstrated that vascularized tissues of large volume and custom geometry can be generated from in vivo bioreactors implanted against the rib periosteum in an ovine model. The effects of different bioreactor scaffold material on tissue ingrowth were measured. To minimize donor site morbidity, tissues generated from bioreactors filled with synthetic graft were transferred as either vascularized free flaps or avascular grafts to a large mandibular defect. It was demonstrated that synthetic graft in an in vivo bioreactor is sufficient to produce free tissue bone flaps capable of integrating with native tissues when transferred to a large mandibular defect in an ovine model.