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Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1 ) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin ( Ctsk )-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk + PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk + PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The periosteum is critical for bone maintenance and healing. However, the in vivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible for lifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration in vivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose : There is a clinical need to better characterize tissue sources being used for stem cell therapies. This study focuses on comparison of cells and connective tissue progenitors (CTPs) derived from native human infrapatellar fatpad (IPFP), synovium (SYN), and periosteum (PERI). Materials and Methods : IPFP, SYN, PERI were harvested from twenty-eight patients undergoing arthroplasty. CTPs were quantitatively characterized using automated colony-forming-unit assay to compare total nucleated cell concentration-[Cell], cells/mg; prevalence-(P CTP ), CTPs/million nucleated cells; CTP concentration-[CTP], CTPs/mg; proliferation and differentiation potential; and correlate outcomes with patient's age and gender. Results : [Cell] did not differ between IPFP, SYN, and PERI. P CTP was influenced by age and gender: patients >60 years, IPFP and SYN had higher P CTP than PERI ( p < 0.001) and females had higher P CTP in IPFP ( p < 0.001) and SYN ( p = 0.001) than PERI. [CTP] was influenced by age: patients <50 years, SYN ( p = 0.0165) and PERI ( p < 0.001) had higher [CTP] than IPFP; patients between 60 and 69 years, SYN ( p < 0.001) had higher [CTP] than PERI; patients >70 years, IPFP ( p = 0.006) had higher [CTP] than PERI. In patients >60 years, proliferation potential of CTPs differed significantly (SYN>IPFP>PERI); however, differentiation potentials were comparable between all three tissue sources. Conclusion : SYN and IPFP may serve as a preferred tissue source for patients >60 years, and PERI along with SYN and IPFP may serve as a preferred tissue source for patients <60 years for cartilage repair. However, the heterogeneity among the CTPs in any given tissue source suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.

Although Prx1 (also known as PRRX1)-expressing cells and their primary cilia are critical for embryonic development, they have yet to be studied in the context of postnatal skeletogenesis owing to the lethality of mouse models. A tamoxifen-inducible Prx1 model has been developed, and we determined that expression directed by this promoter is highly restricted to the cambium layers in the periosteum and perichondrium after birth. To determine the postnatal role of these cambium layer osteochondroprogenitors (CLOPs) and their primary cilia, we developed models to track the fate of CLOPs (Prx1CreER-GFP;Rosa26 tdTomato ) and selectively disrupt their cilia (Prx1CreER-GFP;Ift88 fl/fl ). Our tracking studies revealed that CLOPs populate cortical and trabecular bone, the growth plate and secondary ossification centers during the normal program of postnatal skeletogenesis. Furthermore, animals lacking CLOP cilia exhibit stunted limb growth due to disruptions in endochondral and intramembranous ossification. Histological examination indicates that growth is stunted due to limited differentiation, proliferation and/or abnormal hypertrophic differentiation in the growth plate. Collectively, our results suggest that CLOPs are programmed to rapidly populate distant tissues and produce bone via a primary cilium-mediated mechanism in the postnatal skeleton., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: The fully developed adult skeleton adapts to mechanical forces by generating more bone, usually at the periosteal surface. Progenitor cells in the periosteum are believed to differentiate into bone-forming osteoblasts that contribute to load-induced adult bone formation, but in vivo evidence does not yet exist. Furthermore, the mechanism by which periosteal progenitors might sense physical loading and trigger differentiation is unknown. We propose that periosteal osteochondroprogenitors (OCPs) directly sense mechanical load and differentiate into bone-forming osteoblasts via their primary cilia, mechanosensory organelles known to be involved in osteogenic differentiation., Methods: We generated a diphtheria toxin ablation mouse model and performed ulnar loading and dynamic histomorphometry to quantify the contribution of periosteal OCPs in adult bone formation in vivo. We also generated a primary cilium knockout model and isolated periosteal cells to study the role of the cilium in periosteal OCP mechanosensing in vitro. Experimental groups were compared using one-way analysis of variance or student's t test, and sample size was determined to achieve a minimum power of 80%., Results: Mice without periosteal OCPs had severely attenuated mechanically induced bone formation and lacked the mineralization necessary for daily skeletal maintenance. Our in vitro results demonstrate that OCPs in the periosteum uniquely sense fluid shear and exhibit changes in osteogenic markers consistent with osteoblast differentiation; however, this response is essentially lost when the primary cilium is absent., Conclusions: Combined, our data show that periosteal progenitors are a mechanosensitive cell source that significantly contribute to adult skeletal maintenance. More importantly, an OCP population persists in the adult skeleton and these cells, as well as their cilia, are promising targets for bone regeneration strategies.

Studies on monolayer cultures and whole-animal models for the prediction of the response of native human tissue are associated with limitations. Therefore, more and more laboratories are tending towards multicellular spheroids grown in vitro as a model of native tissues. In addition, they are increasingly used in a wide range of biofabrication methodologies. These 3D microspheroids are generated through a self-assembly process that is still poorly characterised, called cellular aggregation. Here, a system is proposed for the automated, non-invasive and high throughput monitoring of the morphological changes during cell aggregation. Microwell patterned inserts were used for spheroid formation while an automated microscope with 4x bright-field objective captured the morphological changes during this process. Subsequently, the acquired time-lapse images were automatically segmented and several morphological features such as minor axis length, major axis length, roundness, area, perimeter and circularity were extracted for each spheroid. The method was quantitatively validated with respect to manual segmentation on four sets of ± 60 spheroids. The average sensitivities and precisions of the proposed segmentation method ranged from 96.67-97.84% and 96.77-97.73%, respectively. In addition, the different morphological features were validated, obtaining average relative errors between 0.78-4.50%. On average, a spheroid was processed 73 times faster than a human operator. As opposed to existing algorithms, our methodology was not only able to automatically monitor compact spheroids but also the aggregation process of individual spheroids, and this in an accurate and high-throughput manner. In total, the aggregation behaviour of more than 700 individual spheroids was monitored over a duration of 16 hours with a time interval of 5 minutes, and this could be increased up to 48,000 for the described culture format. In conclusion, the proposed system has the potential to be used for unravelling the mechanisms involved in spheroid formation and monitoring their formation during large-scale manufacturing protocols., Competing Interests: The authors have declared that no competing interests exist.

Understanding the role of the PI3K/AKT pathway in regulating basic antler stem cell parameters and angiogenesis may provide an insight into the mechanisms underlying mammalian appendage development. The present study took multiple approaches in vitro to investigate the effects of the PI3K/AKT pathway on antler stem cells. By addition of LY294002, proliferation rate of the antlerogenic periosteum (AP) cells was decreased significantly ( p <0.01), while the proliferation rate of the pedicle periosteum (PP) cells decreased to a lesser extent; the cytoskeleton of the AP cells was essentially collapsed; and the PP cells significantly shrunken. By addition of LY294002 or KU-0063794, formation of networking tubular structures from HUVECs in the AP or PP cell conditioned medium was significantly inhibited; whereas, expression level of VEGF-B mRNA in the AP or PP cells was decreased by the former, and increased by the latter significantly. Therefore, the results suggest that the PI3K/AKT pathway is involved in proliferation and differentiation of the AP and the PP cells, and plays a more important role in the former than in the latter.

Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

Background: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models., Methods: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry., Results: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair., Conclusion: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different locations are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteoblast marker). Comparative gene expression analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and have little overlap with SSCs. This was further supported by the microarray cluster analysis, where the two SSC populations clustered together but are separate from the CD51+ cells. However, when comparing SSC population to controls, we found several genes that are uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.

Annual antler renewal is a stem cell-based epimorphic process driven by antler stem cells (ASCs) resident in antlerogenic periosteum (AP). Antlerogenic periosteal cells express a high level of S100A4, a metastasis-associated protein, which intrigued us to explore what role S100A4 could play in antler regeneration. The present study set out to investigate expression and effects of S100A4 in the ASCs and their progeny. The results showed that not only did cells from the AP express a high level of S100A4, but also the pedicle periosteum and the antler growth center. In the antler growth center, we found S100A4-positive cells were specifically located in blood vessel walls and in vascularized areas. In vitro, recombinant deer S100A4 protein stimulated the proliferation of the AP cells, promoted proliferation, migration and tube formation of human vascular endothelial cells, and enhanced migration of Hela cells, but not AP cells. These findings demonstrated that S100A4 in the ASCs may play a significant role in stimulating angiogenesis, proliferation, but not motility, of ASCs. Deer antlers offer a unique model to explore how rapid cell proliferation with a high level of S100A4 expression is elegantly regulated without becoming cancerous.

Advanced biomaterials that are capable of guiding robust bone regeneration are highly demanded for translational therapy of bone defects or bone augmentation in clinics. One of the strategic approaches is to produce tissue engineering (TE) constructs that mediate bone regeneration by recapitulating the natural bone formation or healing process. In this study, we aimed at producing devitalized mineralized carriers with augmented bone forming capacity via a modified culture protocol (i.e., culture conditions with high calcium and/or phosphate concentrations) that first promotes cell growth and, subsequently, mineralized extracellular matrix (ECM) deposition by human periosteum-derived osteoprogenitor cells (hPDCs) on additive manufactured three-dimensional (3D) porous titanium (Ti)-based scaffolds. Qualitative and quantitative analysis was performed to characterize the physicochemical properties of the produced devitalized mineralized carriers, as well as their effects as carriers on in vitro cell growth and osteochondrogenic differentiation of hPDCs under a perfusion bioreactor culture set-up. The results showed that the modified culture protocol was useful to produce devitalized mineralized carriers with different amount, distribution, composition, and morphology of mineralized matrix that resembled hydroxyapatite, and exhibited different Ca 2+ release kinetics, distinct human bone morphogenetic protein (hBMP)-2, human vascular endothelial growth factor (hVEGF) proteins, and collagen contents. The produced devitalized mineralized carriers supported 3D growth of hPDCs, with minor osteochondrogenic differentiation effects under the perfusion bioreactor culture condition. Subcutaneous implantation of hPDC-seeded devitalized mineralized carriers in athymic nude rats showed nearly five-fold augmentation in the ectopic bone-forming capacity, with no bone induction obtained for unseeded, devitalized mineralized carriers and plain Ti scaffolds. Implantation of devitalized mineralized carriers in critical-sized calvarial defects resulted in encouraging defect bridging as compared with limited defect bridging by plain Ti scaffolds or in empty defects. This defect bridging was not enhanced by implanting hPDC-seeded devitalized mineralized carriers. In conclusion, the investigated modified culture protocol was useful to produce devitalized mineralized carriers with augmented bone-forming capacity, which potentially could aid bone repair or augmentation in clinics.

Tissue engineering based on cell seeding, bioscaffolds, and growth factors has been widely applied for the reconstruction of tissue defects. Recent progress has fueled in vivo tissue engineering techniques in becoming hot topics in regenerative medicine and reconstructive surgery. To improve the efficacy of tissue engineering, we here investigated the roles of cell seeding, bioscaffolds, growth factors, and in vivo microenvironment (IM) in tissue regeneration. Bone marrow-derived stem cells, allogeneic demineralized bone matrix as bioscaffold, and growth factor bone morphogenetic protein 2/transforming growth factor, and the IM of rib periosteum and perichondrium were used in different combinations for the generation of osteochondral composite tissue. Self-regenerated neocomposite tissue based on the IM alone exhibited excellent anatomical configuration, vascularization, biomechanical stability, and function similar to native controls. Our findings indicate that the IM is a crucial factor in biofunctional tissue generation. Further refinement and development of this technique may enable transfer to clinical application with broad spectrum of application.

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects., (© 2014 AlphaMed Press.)

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues.

Elucidation of the periosteum and its regenerative potential has become a hot topic in orthopedics. Yet few review articles address the unique features of periosteum-derived cells, particularly in light of translational therapies and engineering solutions inspired by the periosteum's remarkable regenerative capacity. This review strives to define periosteum-derived cells in light of cumulative research in the field; in addition, it addresses clinical translation of current insights, hurdles to advancement, and open questions in the field. First, we examine the periosteal niche and its inhabitant cells and the key characteristics of these cells in the context of mesenchymal stem cells and their relevance for clinical translation. We compare periosteum-derived cells with those derived from the marrow niche in in vivo studies, addressing commonalities as well as features unique to periosteum cells that make them potentially ideal candidates for clinical application. Thereafter, we review the differentiation and tissue-building properties of periosteum cells in vitro, evaluating their efficacy in comparison with marrow-derived cells. Finally, we address a new concept of banking periosteum and periosteum-derived cells as a novel alternative to currently available autogenic umbilical blood and perinatal tissue sources of stem cells for today's population of aging adults who were "born too early" to bank their own perinatal tissues. Elucidating similarities and differences inherent to multipotent cells from distinct tissue niches and their differentiation and tissue regeneration capacities will facilitate the use of such cells and their translation to regenerative medicine.

Stem cell therapy and cell-based therapies using other progenitor cells are becoming the treatment of choice for many equine orthopedic lesions. Important criteria for obtaining autogenous equine progenitor cells in vitro for use in clinical cell-based therapy include the ability to isolate and expand cells repeatedly to high numbers (millions) required for therapy, in a clinically relevant time frame. Cells must also maintain their ability to differentiate into the tissue type of choice. The objective of this study was to compare isolation and expansion techniques for preparation of periosteal-derived osteogenic progenitor cells for use in commercial autogenous cell-based therapy. Cells were allowed to migrate spontaneously from periosteal tissue or were enzymatically released. Isolated cells were expanded using enzymatic detachment of cells and subsequent monolayer or dynamic culture techniques. Viable osteogenic progenitor cells from each group were counted at 2 weeks, and osteogenic potential determined. Cells isolated or expanded using the explant or bioreactor technique yielded cells at a much lower number per gram of tissue compared with that of enzyme digestion and monolayer expansion, but all cells were able to differentiate into the ostoblast phenotype. Osteogenic progenitor cells isolated by enzymatic release and expanded using monolayer culture reached the highest number of viable cells per gram of donor periosteal tissue while maintaining the ability to differentiate into bone forming cells in vitro. This technique would be an easy, consistent method of preparation of equine osteogenic cells for clinical cell based therapy for orthopedic conditions.

As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers.

An anabolic response driven by osteoblasts is critical for the process of bone healing. Current evidence suggests that these osteoblasts may arise from multiple tissue types and cell lineages. Stem cells present in the bone marrow, periosteum, local soft tissues, vasculature, and/or circulation have been shown to have osteogenic potential. Transplanted cells from these sources have also been shown to incorporate into induced ectopic bone or repaired bone. While these experiments demonstrate the latent capacity of different lineages to assume an osteoblastic phenotype under pro-osteogenic conditions, the actual contribution of the different lineages to various repair situations in vivo remains unclear. This review explores the data arising from different bone formation and repair models. We propose a model suggesting that cells arising from the local tissues, particularly muscle cells, may play an important role in fracture repair under situations where the periosteal and/or bone marrow progenitor populations are depleted.

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering., (Copyright 2007 Wiley Periodicals, Inc.)

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA. We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.