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Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (μ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two μ-dHACM formulations and to investigate the influence of these distributions on the

Reconstruction of large bone defects can be complicated by the presence of both infection and local antibiotic administration. This can be addressed through a two-stage reconstructive approach, called the Masquelet technique, that involves the generation of an induced osteogenic membrane over a temporary poly(methyl methacrylate) (PMMA) space maintainer, followed by definitive reconstruction after the induced membrane is formed. Given that infection and antibiotic delivery each have independent effects on local tissue response, the objective of this study is to evaluate the interaction between local clindamycin release and bacterial contamination with regards to infection prevention and the restoration of pro-osteogenic gene expression in the induced membrane. Porous PMMA space maintainers with or without clindamycin were implanted in an 8 mm rat femoral defect model with or without Staphylococcus aureus inoculation for 28 days in a full-factorial study design (four groups, n = 8/group). Culture results demonstrated that 8/8 animals in the inoculated/no antibiotic group were infected at 4 weeks, which was significantly reduced to 1/8 animals in the inoculated/antibiotic group. Quantitative polymerase chain reaction analysis demonstrated that clindamycin treatment restores inflammatory cytokine and growth factor expression to the same levels as the no inoculation/no antibiotic group, demonstrating that clindamycin can ameliorate the negative effects of bacterial inoculation and does not itself negatively impact the expression of important cytokines. Main effect analysis shows that bacterial inoculation and clindamycin treatment have independent and interacting effects on the gene expression profile of the induced membrane, further highlighting that antibiotics play an important role in the regeneration of infected defects apart from their antimicrobial properties.

In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and -glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31(+), vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31(+)) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

Despite progress in bone tissue engineering, the healing of critically sized diaphyseal defects remains a clinical challenge. A stem cell-based approach is an attractive alternative to current treatment techniques. The objective of this study was to examine the ability of adult stem cells to enhance bone formation when co-delivered with the osteoinductive factor bone morphogenetic protein-2 (BMP-2) in a biologically functionalized hydrogel. First, adipose and bone marrow-derived mesenchymal stem cells (ADSCs and BMMSCs) were screened for their potential to form bone when delivered in an RGD functionalized alginate hydrogel using a subcutaneous implant model. BMMSCs co-delivered with BMP-2 produced significantly more mineralized tissue compared with either ADSCs co-delivered with BMP-2 or acellular hydrogels containing BMP-2. Next, the ability of BMMSCs to heal a critically sized diaphyseal defect with a nonhealing dose of BMP-2 was tested using the alginate hydrogel as an injectable cell carrier. The effect of timing of therapeutic delivery on bone regeneration was also tested in the diaphyseal model. A 7 day delayed injection of the hydrogel into the defect site resulted in less mineralized tissue formation than immediate delivery of the hydrogel. By 12 weeks, BMMSC-loaded hydrogels produced significantly more bone than acellular constructs regardless of immediate or delayed treatment. For immediate delivery, bridging of defects treated with BMMSC-loaded hydrogels occurred at a rate of 75% compared with a 33% bridging rate for acellular-treated defects. No bridging was observed in any of the delayed delivery samples for any of the groups. Therefore, for this cell-based bone tissue engineering approach, immediate delivery of constructs leads to an overall enhanced healing response compared with delayed delivery techniques. Further, these studies demonstrate that co-delivery of adult stem cells, specifically BMMSCs, with BMP-2 enhances bone regeneration in a critically sized femoral segmental defect compared with acellular hydrogels containing BMP-2.

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ε-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

The aim of this study was to establish a critical-sized nonjoint chondral defect animal model and to evaluate its feasibility for testing cartilage regeneration strategies. Dermal biopsy punches 1-4 mm in diameter were used to create cylindrical full-thickness defects in the center of athymic rat xiphoids. The 3 and 4 mm defects remained unhealed 35 days postsurgery, with a large area in the center that had low proteoglycan content based on contrast-enhanced microCT (EPIC-microCT), radiographic, and histological analyses. In a second step, tissue-engineered cartilage was synthesized by culturing primary bovine articular chondrocytes on poly-L-lactic acid (PLA) scaffolds in a perfusion-shear bioreactor for 28 days. These chondrocyte/PLA constructs or primary bovine chondrocytes were implanted into 3-mm-diameter defects. Empty defects and defects implanted with empty PLA scaffolds were used as controls. Xiphoids were harvested 28 days after surgery and examined with faxitron, microCT, and histology using hematoxylin and eosin and safranin-O staining. Both chondrocyte/PLA constructs and chondrocytes alone formed neocartilage. The results indicate that a 3 mm cylindrical defect in a rat xiphoid is an economic, feasible, and reproductive model to evaluate the potential of various constructs for nonjoint cartilage repair.

Over the past 20 years, tissue engineering (TE) has evolved into a thriving research and commercial development field. However, applying TE strategies to musculoskeletal (MSK) and craniofacial tissues has been particularly challenging since these tissues must also transmit loads during activities of daily living. To address this need, organizers invited a small group of bioengineers, surgeons, biologists, and material scientists from academia, industry, and government to participate in a 2½-day conference to develop general and tissue-specific criteria for evaluating new concepts and tissue-engineered constructs, including threshold values of success. Participants were assigned to four breakout groups representing commonly injured tissues, including tendon and ligament, articular cartilage, meniscus and temporomandibular joint, and bone and intervertebral disc. Working in multidisciplinary teams, participants first carefully defined one or two important unmet clinical needs for each tissue type, including current standards of care and the potential impact of TE solutions. The groups then sought to identify important parameters for evaluating repair outcomes in preclinical studies and to specify minimally acceptable values for these parameters. The importance of in vitro TE studies was then discussed in the context of these preclinical studies. Where data were not currently available from clinical, preclinical, or culture studies, the groups sought to identify important areas of preclinical research needed to speed the development process. This report summarizes the findings of the conference.

Severe birth defects occur in ∼ 2-3% of live-born infants and are a leading cause of death in the young. Structural malformations can occur in just about any major organ system and often their causes are unknown. The pediatric population presents a unique set of opportunities to the field of tissue engineering and regenerative medicine (TERM). Infants and young children have significantly greater regenerative capacity than adults, which could be leveraged in TERM strategies. Children also arguably stand to benefit the most from TERM. Although the lack of growth potential and relatively short life span of synthetic materials may be suitable for adults, it is unacceptable for children. Furthermore, given that there is a particular scarcity of pediatric donor organs, the need for living functional tissue replacements that can grow with the child is quite evident. There is enormous potential for the TERM community to address the needs of the pediatric population.