Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes.

Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy.

Emerging drugs for the treatment of wound healing.

INTRODUCTION: Wound healing can be characterized as underhealing, as in the setting of chronic wounds, or overhealing, occurring with hypertrophic scar formation after burn injury. Topical therapies targeting specific biochemical and molecular pathways represent a promising avenue for improving and, in some cases normalizing, the healing process. AREAS COVERED: A brief overview of both normal and pathological wound healing has been provided, along with a review of the current clinical guidelines and treatment modalities for chronic wounds, burn wounds and scar formation. Next, the major avenues for wound healing drugs, along with drugs currently in development, are discussed. Finally, potential challenges to further drug development, and future research directions are discussed. EXPERT OPINION: The large body of research concerning wound healing pathophysiology has provided multiple targets for topical therapies. Growth factor therapies with the ability to be targeted for localized release in the wound microenvironment are most promising, particularly when they modulate processes in the proliferative phase of wound healing.

Pharmacological rescue of diabetic skeletal stem cell niches.

Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.

Small Molecule Inhibition of Transforming Growth Factor Beta Signaling Enables the Endogenous Regenerative Potential of the Mammalian Calvarium.

Current approaches for the treatment of skeletal defects are suboptimal, principally because the ability of bone to repair and regenerate is poor. Although the promise of effective cellular therapies for skeletal repair is encouraging, these approaches are limited by the risks of infection, cellular contamination, and tumorigenicity. Development of a pharmacological approach would therefore help avoid some of these potential risks. This study identifies transforming growth factor beta (TGFß) signaling as a potential pathway for pharmacological modulation in vivo. We demonstrate that inhibition of TGFß signaling by the small molecule SB431542 potentiates calvarial skeletal repair through activation of bone morphogenetic protein (BMP) signaling on osteoblasts and dura mater cells participating in healing of calvarial defects. Cells respond to inhibition of TGFß signaling by producing higher levels of BMP2 that upregulates inhibitory Smad6 expression, thus providing a negative feedback loop to contain excessive BMP signaling. Importantly, study on human osteoblasts indicates that molecular mechanism(s) triggered by SB431542 are conserved. Collectively, these data provide insights into the use of small molecules to modulate key signaling pathways for repairing skeletal defects.

A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield.

BACKGROUND: The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol. METHODS: Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, the authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test the ability of the isolated cells to enhance adipogenesis, the cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning. RESULTS: The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with the conventional method. Cells harvested using the new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with the conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice. CONCLUSIONS: The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis.

BACKGROUND: Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. METHODS: Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. RESULTS: In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. CONCLUSIONS: BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.

Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies.

Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

RNA Sequencing for Identification of Differentially Expressed Noncoding Transcripts during Adipogenic Differentiation of Adipose-Derived Stromal Cells.

BACKGROUND: Adipose-derived stromal cells represent a relatively abundant source of multipotent cells, with many potential applications in regenerative medicine. The present study sought to demonstrate the use of RNA sequencing in identifying differentially expressed transcripts, particularly long noncoding RNAs, associated with adipogenic differentiation to gain a clearer picture of the mechanisms responsible for directing adipose-derived stromal cell fate toward the adipogenic lineage. METHODS: Human adipose-derived stromal cells were cultured in adipogenic differentiation media, and RNA was harvested at days 0, 1, 3, 5, and 7. Directional RNA sequencing libraries were prepared and sequenced. Paired-end reads were mapped to the human genome reference sequence hg19. Transcriptome assembly was performed and significantly differentially expressed transcripts were identified. Gene ontology term analysis was then performed to identify coding and noncoding transcripts of interest. Differential expression was verified by quantitative real-time polymerase chain reaction. RESULTS: Of 2868 significantly differentially expressed transcripts identified, 207 were noncoding. Enriched gene ontology terms among up-regulated coding transcripts notably reflected differentiation toward the adipogenic lineage. Enriched gene ontology terms among down-regulated coding transcripts reflected growth arrest. Guilt-by-association analysis revealed noncoding RNA candidates with potential roles in the process of adipogenic differentiation. CONCLUSIONS: The precise mechanisms that guide lineage-specific differentiation in multipotent cells are not yet fully understood. Defining long noncoding RNAs associated with adipogenic differentiation allows for potential manipulation of regulatory pathways in novel ways. The authors present RNA sequencing as a powerful tool for expanding the understanding of adipose-derived stromal cells and developing novel applications within regenerative medicine.

Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent.

BACKGROUND: Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. METHODS: Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 µl of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. RESULTS: Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 µl of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone CONCLUSIONS: : The authors' study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.

Studies in fat grafting: Part IV. Adipose-derived stromal cell gene expression in cell-assisted lipotransfer.

BACKGROUND: Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting. METHODS: Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells. RESULTS: Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein-positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting. CONCLUSIONS: Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.

Isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

Assessment of viability of human fat injection into nude mice with micro-computed tomography.

Lipotransfer is a vital tool in the surgeon's armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.(1) However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. (1-3) To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes. Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells.

Adipose tissue contains an abundant source of multipotent mesenchymal cells termed "adipose-derived stromal cells" (ASCs) that hold potential for regenerative medicine. However, the heterogeneity inherent to ASCs harvested using standard methodologies remains largely undefined, particularly in regards to differences across donors. Identifying the subpopulations of ASCs predisposed toward differentiation along distinct lineages holds value for improving graft survival, predictability, and efficiency. Human ASCs (hASCs) from three different donors were independently isolated by density-based centrifugation from adipose tissue and maintained in culture or differentiated along either adipogenic or osteogenic lineages using differentiation media. Undifferentiated and differentiated hASCs were then analyzed for the presence of 242 human surface markers by flow cytometry analysis. By comprehensively characterizing the surface marker profile of undifferentiated hASCs using flow cytometry, we gained novel insights into the heterogeneity underlying protein expression on the surface of cultured undifferentiated hASCs across different donors. Comparison of the surface marker profile of undifferentiated hASCs with hASCs that have undergone osteogenic or adipogenic differentiation allowed for the identification of surface markers that were upregulated and downregulated by osteogenic or adipogenic differentiation. Osteogenic differentiation induced upregulation of CD164 and downregulation of CD49a, CD49b, CD49c, CD49d, CD55, CD58, CD105, and CD166 while adipogenic differentiation induced upregulation of CD36, CD40, CD146, CD164, and CD271 and downregulation of CD49b, CD49c, CD49d, CD71, CD105, and CD166. These results lend support to the notion that hASCs isolated using standard methodologies represent a heterogeneous population and serve as a foundation for future studies seeking to maximize their regenerative potential through fluorescence-activated cell sorting-based selection before therapy.

Studies in fat grafting: Part III. Fat grafting irradiated tissue--improved skin quality and decreased fat graft retention.

BACKGROUND: Following radiation therapy, skin becomes fibrotic and can present a difficult problem for reconstructive surgeons. There is an increasing belief that fat grafting under irradiated skin can reverse the damage caused by radiation. The present study evaluated the effect of fat grafting on irradiated skin, along with fat graft quality and retention rates in irradiated tissue. METHODS: Nine adult Crl:NU-Foxn1 CD-1 mice underwent 30-Gy external beam irradiation of the scalp. Four weeks after irradiation, scalp skin from irradiated and nonirradiated mice was harvested and compared histologically for dermal thickness, collagen content, and vascular density. Human fat grafts were then injected in the subcutaneous plane of the scalp. Skin assessment was performed in the irradiated group at 2 and 8 weeks after grafting, and fat graft retention was measured at baseline and every 2 weeks up to 8 weeks after grafting using micro-computed tomography. Finally, fat graft samples were explanted at 8 weeks, and quality scoring was performed. RESULTS: Fat grafting resulted in decreased dermal thickness, decreased collagen content, and increased vascular density in irradiated skin. Computed tomographic analysis revealed significantly decreased fat graft survival in the irradiated group compared with the nonirradiated group. Histologic scoring of explanted fat grafts demonstrated no difference in quality between the irradiated and nonirradiated groups. CONCLUSIONS: Fat grafting attenuates dermal collagen deposition and vessel depletion characteristic of radiation fibrosis. Although fat graft retention rates are significantly lower in irradiated than in nonirradiated tissue, the quality of retained fat between the groups is similar.

Capillary force seeding of hydrogels for adipose-derived stem cell delivery in wounds.

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage.

BACKGROUND: Adipose tissue represents an abundant and easily accessible source of multipotent cells that may serve as an excellent building block for tissue engineering. However, adipose-derived stromal cells (ASCs) are a heterogeneous group and subpopulations may be identified with enhanced osteogenic potential. METHODS: Human ASC subpopulations were prospectively isolated based on expression of bone morphogenetic protein receptor type-IB (BMPR-IB). Unsorted, BMPR-IB(+), and BMPR-IB(-) cells were analyzed for their osteogenic capacity through histological staining and gene expression. To evaluate their in vivo osteogenic potential, critical-sized calvarial defects were created in immunocompromised mice and treated with unsorted, BMPR-IB(+), or BMPR-IB(-) cells. Healing was assessed using microcomputed tomography and pentachrome staining of specimens at 8 weeks. RESULTS: Increased osteogenic differentiation was noted in the BMPR-IB(+) subpopulation, as demonstrated by alkaline phosphatase staining at day 7 and extracellular matrix mineralization with Alizarin red staining at day 14. This was also associated with increased expression for osteocalcin, a late marker of osteogenesis. Radiographic analysis demonstrated significantly enhanced healing of critical-sized calvarial defects treated with BMPR-IB(+) ASCs compared with unsorted or BMPR-IB(-) cells. This was confirmed through pentachrome staining, which revealed more robust bone regeneration in the BMPR-IB(+) group. CONCLUSION: BMPR-IB(+) human ASCs have an enhanced ability to form bone both in vitro and in vivo. These data suggest that positive selection for BMPR-IB(+) and manipulation of the BMP pathway in these cells may yield a highly osteogenic subpopulation of cells for bone tissue engineering.

The Role of Hypoxia-Inducible Factor in Wound Healing.

Significance: Poor wound healing remains a significant health issue for a large number of patients in the United States. The physiologic response to local wound hypoxia plays a critical role in determining the success of the normal healing process. Hypoxia-inducible factor-1 (HIF-1), as the master regulator of oxygen homeostasis, is an important determinant of healing outcomes. HIF-1 contributes to all stages of wound healing through its role in cell migration, cell survival under hypoxic conditions, cell division, growth factor release, and matrix synthesis throughout the healing process. Recent Advances: Positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic ischemic wound closure and are currently undergoing clinical trials for treatment of several human-ischemia-based conditions. Critical Issues: HIF-1 deficiency and subsequent failure to respond to hypoxic stimuli leads to chronic hypoxia, which has been shown to contribute to the formation of nonhealing ulcers. In contrast, overexpression of HIF-1 has been implicated in fibrotic disease through its role in increasing myofibroblast differentiation leading to excessive matrix production and deposition. Both positive and negative regulators of HIF-1 therefore provide important therapeutic targets that can be used to manipulate HIF-1 expression where an excess or deficiency in HIF-1 is known to correlate with pathogenesis. Future Directions: Targeting HIF-1 during wound healing has many important clinical implications for tissue repair. Counteracting the detrimental effects of excessive or deficient HIF-1 signaling by modulating HIF-1 expression may improve future management of poorly healing wounds.

Osteoclast derivation from mouse bone marrow.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.