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While formation and regeneration of the skeleton have been studied for a long period of time, significant scientific advances in this field continue to emerge based on an unmet clinical need to improve options to promote bone repair. In this review, we discuss the relationship between mechanisms of bone formation and bone regeneration. Data clearly show that regeneration is not simply a reinduction of the molecular and cellular programs that were used for development. Instead, the mechanical environment exerts a strong influence on the mode of repair, while during development, cell-intrinsic processes drive the mode of skeletal formation. A major advance in the field has shown that cell fate is flexible, rather than terminal, and that chondrocytes are able to differentiate into osteoblasts and other cell types during development and regeneration. This is discussed in a larger context of regeneration in vertebrates as well as the clinical implication that this shift in understanding presents.

Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding β-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated β-catenin GOF mRNA encapsulated in SM-102 LNPs (SM-102-β-catenin GOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2 . When testing SM-102-β-catenin GOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 μg concentration at 2 weeks post-fracture. μCT testing confirmed that SM-102-β-catenin GOF mRNA promoted bone formation in vivo , revealing significantly more bone volume over total volume in the 45 μg group. Thus, we generated a novel mRNA-based therapeutic encoding a β-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery., Competing Interests: Anna Laura Nelson, Chiara Mancino, Xueqin Gao, Josh Choe, Laura Chubb, Katherine Williams, Molly Czachor, Ralph Marcucio and Francesca Taraballi have nothing to disclose. Dr. Johnny Huard discloses an unpaid position on the leadership for Orthopaedic Research Society (ORS). JH discloses royalties from Cook Myosite, Inc. Dr. Cooke is on the Scientific Advisory Board of Humann Inc, which makes products related to nitric oxide and cardiovascular health; is an inventor on multiple patents assigned to Stanford University or Houston Methodist Hospital related to endothelial function and regeneration; and has been a collaborator and co-author with Dr. Zhen Chen. Dr. Chelsea Bahney discloses an unpaid position on the leadership for Orthopaedic Research Society (ORS), Tissue Engineering and Regenerative Medicine International Society (TERMIS), and the Orthopaedic Trauma Association (OTA). CB also discloses IP royalties from Iota Biosciences, Inc. for US Patent 041263 and a Review Editor role for Frontiers in Skeletal Physiology. Dr. Nicole Ehrhart discloses paid consultant positions for Onkos Surgical Inc. and Ripple Neuromed Inc. ALN, CM, JH, RM, JCooke, FT, CB, NE also disclose inventorship on PCT/US2022 Application No. 63/155,263, entitled MCM For Gene Therapy to Activate Wnt Pathway. These entities provided no funding for this research and there are no conflicts of interest with the work presented in this manuscript. Anna Laura Nelson, Dr. Johnny Huard, Dr. Xueqin Gao, Molly Czachor and Dr. Chelsea Bahney are all paid employees of the non-profit Steadman Philippon Research Institute (SPRI). SPRI exercises special care to identify any financial interests or relationships related to research conducted here. During the past calendar year, SPRI has received grant funding or in-kind donations from Arthrex, DJO, MLB, Ossur, Siemens, Smith & Nephew, XTRE, and philanthropy. These funding sources provided no support for the work presented in this manuscript unless otherwise noted., (© 2024 The Authors.)

Mesenchymal stromal cells (MSCs) are a promising cell population for musculoskeletal cell-based therapies due to their multipotent differentiation capacity and complex secretome. Cells from younger donors are mechanosensitive, evidenced by changes in cell morphology, adhesivity, and differentiation as a function of substrate stiffness in both two- and three-dimensional culture. However, MSCs from older individuals exhibit reduced differentiation potential and increased senescence, limiting their potential for autologous use. While substrate stiffness is known to modulate cell phenotype, the influence of the mechanical environment on senescent MSCs is poorly described. To address this question, we cultured irradiation induced premature senescent MSCs on polyacrylamide hydrogels and assessed expression of senescent markers, cell morphology, and secretion of inflammatory cytokines. Compared to cells on tissue culture plastic, senescent MSCs exhibited decreased markers of the senescence associated secretory phenotype (SASP) when cultured on 50 kPa gels, yet common markers of senescence (e.g., p21, CDKN2A, CDKN1A) were unaffected. These effects were muted in a physiologically relevant heterotypic mix of healthy and senescent MSCs. Conditioned media from senescent MSCs on compliant substrates increased osteoblast mineralization compared to conditioned media from cells on TCP. Mixed populations of senescent and healthy cells induced similar levels of osteoblast mineralization compared to healthy MSCs, further indicating an attenuation of the senescent phenotype in heterotypic populations. These data indicate that senescent MSCs exhibit a decrease in senescent phenotype when cultured on compliant substrates, which may be leveraged to improve autologous cell therapies for older donors., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro . When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1β and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo . Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nelson, Fontana, Chubb, Choe, Williams, Regan, Huard, Murphy, Ehrhart and Bahney.)

Background: Quercetin, a natural flavonoid, has shown promise as a senolytic agent for various degenerative diseases. Recently, its protective effect against osteoarthritis (OA), a representative age-related disease of the musculoskeletal system, has attracted much attention. The aim of this study is to summarize and analyze the current literature on the effects of quercetin on OA cartilage in in vivo preclinical studies., Methods: The Medline (via/using PubMed), Embase, and Web of Science databases were searched up to March 10th, 2023. Risk of bias and the qualitative assessment including mechanisms of all eligible studies and a meta-analysis of cartilage histological scores among the applicable studies was performed., Results: A total of 12 in vivo animal studies were included in this systematic review. A random-effects meta-analysis was performed on six studies using the Osteoarthritis Research Society International (OARSI) scoring system, revealing that quercetin significantly improved OA cartilage OARSI scores (SMD, -6.30 [95% CI, -9.59 to -3.01]; P = 0.0002; heterogeneity: I2 = 86%). The remaining six studies all supported quercetin's protective effects against OA during disease and aging., Conclusions: Quercetin has shown beneficial effects on cartilage during OA across animal species. Future double-blind randomized controlled clinical trials are needed to verify the efficacy of quercetin in the treatment of OA in humans., Competing Interests: Conflict of interest The authors report the following potential conflicts of interest or sources of funding: Steadman Philippon Research Institute has received grant funding or in-kind donations from Arthrex, DJO, MLB, Ossur, Siemens, Smith & Nephew, and XTRE. JH discloses an unpaid position on the leadership for Orthopaedic Research Society (ORS) and royalties from Cook Myosite. M.J.P. receives support from Smith & Nephew, Linvatec, Bledsoe, DonJoy, Arthrosurface, MIS, Siemens, Arthrex, National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Disease, and National Institute of Aging; and owns stock or stock options in Arthrosurface, MJP Innovations, MIS, and Vail Valley Surgery Center. The other authors have no conflicts of interest to declare. CB discloses unpaid leadership positions for ORS, Tissue Engineering and Regenerative Medicine International Society (TERMIS), and the Orthopaedic Trauma Association (OTA). CB also discloses IP royalties from Iota Biosciences, Inc. for US Patent 041263 and a Review Editor role for Frontiers in Skeletal Physiology., (Copyright © 2023. Published by Elsevier B.V.)

Background: Senescence, a characteristic of cellular aging and inflammation, has been linked to the acceleration of osteoarthritis. The purpose of this study is to prospectively identify, measure, and compare senescent profiles in synovial fluid and peripheral blood in patients with an acute knee injury within 48 h., Methods: Seven subjects, aged 18-60 years, with an acute ACL tear with effusion were prospectively enrolled. Synovial fluid and peripheral blood samples were collected and analyzed by flow cytometry, using senescent markers C12FDG and CD87. The senescent versus pro-regenerative phenotype was probed at a gene and protein level using qRT-PCR and multiplex immunoassays., Results: C 12 FDG and CD87 positive senescent cells were detected in the synovial fluid and peripheral blood of all patients. Pro-inflammatory IL-1β gene expression measured in synovial fluid was significantly higher ( p = 0.0156) than systemic/blood expression. Senescent-associated factor MMP-3 and regenerative factor TIMP-2 were significantly higher in synovial fluid compared to blood serum. Senescent-associated factor MMP-9 and regenerative factor TGFβ-2 were significantly elevated in serum compared to synovial fluid. Correlation analysis revealed that C12FDG ++ /CD87 ++ senescent cells in synovial fluid positively correlated with age-related growth-regulated-oncogene (ρ = 1.00, p < 0.001), IFNγ (ρ = 1.00, p < 0.001), IL-8 (ρ = 0.90, p = 0.0374), and gene marker p16 (ρ = 0.83, p = 0.0416)., Conclusions: There is an abundance of senescent cells locally and systemically after an acute ACL tear without a significant difference between those present in peripheral blood compared to synovial fluid. This preliminary data may have a role in identifying strategies to modify the acute environment within the synovial fluid, either at the time of acute ligament injury or reconstruction surgery.

Aging leads to several geriatric conditions including osteoporosis (OP) and associated frailty syndrome. Treatments for these conditions are limited and none target fundamental drivers of pathology, and thus identifying strategies to delay progressive loss of tissue homeostasis and functional reserve will significantly improve quality of life in elderly individuals. A fundamental property of aging is the accumulation of senescent cells. Senescence is a cell state defined by loss of proliferative capacity, resistance to apoptosis, and the release of a proinflammatory and anti-regenerative senescence-associated secretory phenotype (SASP). The accumulation of senescent cells and SASP factors is thought to significantly contribute to systemic aging. Senolytics-compounds which selectively target and kill senescent cells-have been characterized to target and inhibit anti-apoptotic pathways that are upregulated during senescence, which can elicit apoptosis in senescent cells and relieve SASP production. Senescent cells have been linked to several age-related pathologies including bone density loss and osteoarthritis in mice. Previous studies in murine models of OP have demonstrated that targeting senescent cells pharmacologically with senolytic drugs can reduce symptomology of the disease. Here, we demonstrate the efficacy of senolytic drugs (dasatinib, quercetin, and fisetin) to improve age-associated degeneration in bone using the Zmpste24 -/- (Z24 -/- ) progeria murine system for Hutchinson-Gilford progeria syndrome (HGPS). We found that the combination of dasatinib plus quercetin could not significantly mitigate trabecular bone loss although fisetin administration could reduce bone density loss in the accelerated aging Z24 -/- model. Furthermore, the overt bone density loss observed in the Z24 -/- model reported herein highlights the Z24 model as a translational model to recapitulate alterations in bone density associated with advanced age. Consistent with the "geroscience hypothesis," these data demonstrate the utility of targeting a fundamental driver of systemic aging (senescent cell accumulation) to alleviate a common condition with age, bone deterioration., Competing Interests: The authors declare that they have no conflicts of interest relative to the current study. Drs. Hambright and Huard declare inventorship on US PCT Application 16994356 “Methods for treating disease associated with senescence.” Dr. Bahney discloses IP royalties from Iota Biosciences, Inc., for US Patent 041263 and an Associate Editor role for the Journal of Tissue Engineering and Regenerative Medicine (JTERM). Dr. Bahney also discloses leadership roles for the Orthopaedic Research Society, Orthopaedic Trauma Association, and Tissue Engineering and Regenerative Medicine Society. Many authors are employees of Steadman Philippon Research Institute which has received institutional grant funding or in-kind donations from Smith & Nephew, Arthrex, Stryker, Ossur, SubioMed, DJO, Wright Medical, Canon, Icarus Medical, and Medtronic., (Copyright © 2023 William S. Hambright et al.)

Introduction: Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair., Methods: Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury., Results: We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers., Discussion: Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Haffner-Luntzer, Weber, Morioka, Lackner, Fischer, Bahney, Ignatius, Kalbitz, Marcucio and Miclau.)

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fok, Gresham, Ryan, Osipov, Bahney and Leach.)

Activation of the canonical Wingless-related integration site (Wnt) pathway has been shown to increase bone formation and therefore has therapeutic potential for use in orthopedic conditions. However, attempts at developing an effective strategy to achieve Wnt activation has been met with several challenges. The inherent hydrophobicity of Wnt ligands makes isolating and purifying the protein difficult. To circumvent these challenges, many have sought to target extracellular inhibitors of the Wnt pathway, such as Wnt signaling pathway inhibitors Sclerostin and Dickkopf-1, or to use small molecules, ions and proteins to increase target Wnt genes. Here, we review systemic and localized bioactive approaches to enhance bone formation or improve bone repair through antibody-based therapeutics, synthetic Wnt surrogates and scaffold doping to target canonical Wnt. We conclude with a brief review of emerging technologies, such as mRNA therapy and Clustered Regularly Interspaced Short Palindromic Repeats technology, which serve as promising approaches for future clinical translation., (© 2022 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

As the worldwide population progresses in age, there is an increasing need for effective treatments for age-associated musculoskeletal conditions such as osteoporosis and osteoarthritis (OA). Fisetin, a natural flavonoid, has garnered attention as a promising pharmaceutical option for treating or delaying the progression of osteoporosis and OA. However, there is no systematic review of the effects of fisetin on bone and cartilage. The aim of this review is to report the latest evidence on the effects of fisetin on bone and cartilage, with a focus on clinical significance. The PubMed, Embase, and Cochrane Library databases were searched up to December 9th 2021 to evaluate the effects of fisetin on bone and cartilage in in vitro studies and in vivo preclinical animal studies. The risk of bias, quality, study design, sample characteristics, dose and duration of fisetin treatment, and outcomes of the 13 eligible studies were analyzed in this systematic review. Qualitative evaluation was conducted for each study due to differences in animal species, cell type, created disease model, dose and duration of fisetin treatment, and time between intervention and assessment among the eligible studies. The beneficial effects of fisetin on osteoporosis have been demonstrated in in vitro and in vivo preclinical studies across animal species. Similarly, the beneficial effects of fisetin on OA have been demonstrated in in vivo preclinical animal studies, but the reports on OA are still limited. Fisetin, a natural supplement can be use in orthobiologics treatment, as adjuvant to orthopaedic surgery, to improve clinical outcome., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

While most bones fully self-heal, certain diseases require bone allograft to assist with fracture healing. Bone allografts offer promise as treatments for such fractures due to their osteogenic properties. However, current bone allografts made of decellularized bone extracellular matrix (ECM) have high failure rates, and thus grafts which improve fracture healing outcomes are needed. Understanding specific changes to the ECM proteome during normal fracture healing would enable the identification of key proteins that could be used enhance osteogenicity of bone allograft. Here, we performed a timeseries analysis of the fracture callus in mice to investigate proteomic and mineralization changes to the ECM at key stages of fracture healing. We found that changes to the ECM proteome largely coincide with the distinct phases of fracture healing. Basement membrane proteins (AGRN, COL4, LAMA), cartilage proteins (COL2A1, ACAN), and collagen crosslinking enzymes (LOXL, PLOD, ITIH) were initially upregulated, followed by bone specific proteoglycans and collagens (IBSP, COL1A1). Various tissue proteases (MMP2, 9, 13, 14; CTSK, CTSG, ELANE) were expressed at different levels throughout fracture healing. These changes coordinated with mineralization of the fracture callus, which increased steeply during the initial stages of healing. Interestingly the later timepoint was characterized by a response to wound healing and high expression of clotting factors (F2, 7, 9, 10). We identified ELANE and ITIH2 as tissue remodeling enzymes having no prior known involvement with fracture healing. This data can be further mined to identify regenerative proteins for enhanced bone graft design.

Background: Meniscal vascular supply is an important determinant of its healing potential. It has been reported that only the peripheral 30% of the meniscus is vascularized in cadavers aged 53 to 94 years; however, the vascularity in young patients, in whom meniscal repair is more often performed, is unknown., Purpose: The primary objective was to analyze and measure the microvascular anatomy of the meniscus in adult cadaveric specimens <35 years old. The secondary objective was to assess angiogenic potential by quantifying regional gene expression in a meniscal allograft cohort <45 years old., Study Design: Descriptive laboratory study., Methods: In part 1 of this study, 13 fresh-frozen cadaveric knees (age range, 22-34 years; mean, 28.5 years) underwent popliteal artery India ink injection and tissue clearing using a Spalteholz technique, followed by microvascular vascular measurement. In part 2, mRNA was isolated from 13 meniscal allografts (age range, 17-43 years; mean, 27.2 years), and expression of angiogenic genes, vascular endothelial growth factor ( VEGF ), and vascular endothelial growth factor receptor 1 ( FLT1 ) was quantified using real-time polymerase chain reaction., Results: The maximal depth of vascular penetration into the periphery of the medial and lateral menisci ranged from 0% to 42% and 0% to 48%, respectively. There was variation in the degree of vascular penetration within the medial meniscus, with the posterior horn having a significantly smaller depth of penetration (median, 8.7%) than that of the anterior horn (median, 17.4%; P < .0001) or midbody (median, 17.5%; P = .0003). There were no differences in angiogenesis gene expression ( VEGF / FLT1 ) based on circumferential or radial meniscal locations., Conclusion: The vascular supply of the medial and lateral menisci in specimens from adults <35 years of age extended farther than what was reported in specimens from older individuals; however, median values remained consistent. Gene expression of the angiogenic marker VEGF was low throughout all regions of uninjured menisci from young adults, which is consistent with reports in older specimens., Clinical Relevance: Improved understanding of meniscal vascular supply in young adults is critical to informing clinical treatment decisions.

Despite its importance as a key parameter of child health and development, growth velocity is difficult to determine in real time because skeletal growth is slow and clinical tools to accurately detect very small increments of growth do not exist. We report discovery of a marker for skeletal growth in infants and children. The intact trimeric noncollagenous 1 (NC1) domain of type X collagen, the marker we designated as CXM for Collagen X Marker, is a degradation by-product of endochondral ossification that is released into the circulation in proportion to overall growth plate activity. This marker corresponds to the rate of linear bone growth at time of measurement. Serum concentrations of CXM plotted against age show a pattern similar to well-established height growth velocity curves and correlate with height growth velocity calculated from incremental height measurements in this study. The CXM marker is stable once collected and can be accurately assayed in serum, plasma, and dried blood spots. CXM testing may be useful for monitoring growth in the pediatric population, especially responses of infants and children with genetic and acquired growth disorders to interventions that target the underlying growth disturbances. The utility of CXM may potentially extend to managing other conditions such as fracture healing, scoliosis, arthritis, or cancer., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This review focuses on current tissue engineering strategies for promoting vascularized bone regeneration. We review the role of angiogenic growth factors in promoting vascularized bone regeneration and discuss the different therapeutic strategies for controlled/sustained growth factor delivery. Next, we address the therapeutic uses of stem cells in vascularized bone regeneration. Specifically, this review addresses the concept of co-culture using osteogenic and vasculogenic stem cells, and how adipose derived stem cells compare to bone marrow derived mesenchymal stem cells in the promotion of angiogenesis. We conclude this review with a discussion of a novel approach to bone regeneration through a cartilage intermediate, and discuss why it has the potential to be more effective than traditional bone grafting methods., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fracture healing is a unique multifaceted process requiring the presence of cells, molecular mediators, and angiogenic factors. The state of inflammation dominates the initial phase, but the ideal magnitude and duration of the process for an optimal outcome remains obscure. Biological response modifiers, such as platelet-rich plasma (PRP) preparations, have been used to reconstitute the desirable early inflammatory state, but the results obtained remain inconclusive. Ongoing research to characterize and quantify the inflammatory response after bone fracture is essential in order to better understand the molecular insights of this localized reaction and to expand our armamentarium in the management of patients with an impaired fracture healing response. Non-steroidal anti-inflammatory drugs frequently administered for analgesia after trauma procedures continue to be a cause of concern for a successful bone repair response.

Stem cells offer great promise to help understand the normal mechanisms of tissue renewal, regeneration, and repair, and also for development of cell-based therapies to treat patients after tissue injury. Most adult tissues contain stem cells and progenitor cells that contribute to homeostasis, remodeling, and repair. Multiple stem and progenitor cell populations in bone are found in the marrow, the endosteum, and the periosteum. They contribute to the fracture healing process after injury and are an important component in tissue engineering approaches for bone repair. This review focuses on current concepts in stem cell biology related to fracture healing and bone tissue regeneration, as well as current strategies and limitations for clinical cell-based therapies.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice, after application of bioactive molecules, after altering physiological parameters of healing, after modifying the amount or time of stabilization, after distraction osteogenesis, after creation of a non-union, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

Biological activity can be added to synthetic scaffolds by incorporating functional peptide sequences that provide enzyme-mediated degradation sites, facilitate cellular adhesion or stimulate signaling pathways. Poly(ethylene glycol) diacrylate is a popular synthetic base for tissue engineering scaffolds because it creates a hydrophilic environment that can be chemically manipulated to add this biological functionality. Furthermore, the acrylate groups allow for encapsulation of cells using photopolymerization under physiological conditions. One complication with the addition of these peptides is that aromatic amino acids absorb light at 285 nm and compete with the ultraviolet (UV)-sensitive photoinitiators such as IrgacureTM 2959 (I2959), the most commonly used initiator for cytocompatible photoencapsulation of cells into synthetic scaffolds. In this study we define non-toxic conditions for photoencapsulation of human mesenchymal stem cells (hMSC) in PEGDA scaffolds using a visible light photoinitiator system composed of eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone. This visible light photoinitiator produced hydrogel scaffolds with an increased viability of encapsulated hMSCs and a more tightly crosslinked network in one-third the time of UV polymerization with I2959.