We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells. Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis

Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone

Introduction. Excessive bone mass and microarchitecture loss exacerbate the risk of osteoporotic fracture, a skeletal disorder attributable to disability in the elder. Excessive marrow adipose development at the expense of osteoblastic bone acquisition is a prominent feature of aging-induced osteoporotic skeletons. MicroRNA-29a (miR-29a) modulates osteogenic and adipogenic commitment of mesenchymal progenitor cells. The purposes of this study were to test if miR-29a overexpression changed bone mass or microstructure in aged skeletal tissues. Materials/Methods. Transgenic mice that overexpressed miR-29a in osteoblasts driven by osteocalcin promoter (miR-29aTg) were generated. Littermates without carrying construct of interest were used as wild-type mice (WT). 3- and 12-month-old mice were designated into young and aged groups respectively. Bone mineral density (BMD), cortical, trabecular microarchitecture and morphometric profiles were quantified with ultrahigh resolution μCT system. Primary bone-marrow mesenchymal stem cells (BMMSCs) were incubated in osteogenic and adipogenic conditions. Expressions of osteogenic and adipogenic marker were quantified with RT-PCR. Results. Skeletons in the aged WT group showed 65% decrease in BMD in association with 72% reduction in miR-29a expression and 2.3-fold elevation in marrow fat volume as compared with those in young WT group. The young miR-29aTg mice showed 35–48% increases in serum osteocalcin and bone alkaline phosphatase levels concomitant with 22–35% increases in BMD, trabecular BV/TV, Tb.Th, Tb.N, and cortical morphology than those of young WT mice. Intriguing analyses are that miR-29aTg mice exhibited mild responses to the aging provocation of BMD loss, trabecular, cortical microstructure deterioration, and fatty marrow histopathology. In vitro, primary BMMSCs in miR-29aTg mice showed significant increases in osteogenic gene expression and mineralized matrices as probed with von Kossa staining, whereas adipogenic gene expression and adipocyte formation were evidently reduced as evidenced by fluorescence Nile Red. Conclusion. miR-29a overexpression in osteoblasts facilitates skeletal tissue anabolism. High osteogenic lineage commitment of bone-marrow mesenchymal progenitor cells contributes to high bone mass and microstructure phenotypes promoted by miR-29a signaling. Analyses shed a new light on the miR-29a signaling protection against the aging escalation of osteoporosis pathogenesis

Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications

Background. Long-term glucocorticoid treatment increases incidence of osteoporotic or osteonecrotic disorders. Excessive bone loss and marrow fat accumulation are prominent features of glucocorticoid-induced osteoporosis. MicroRNA-29 (miR-29) family members reportedly modulate lineage commitment of stem cells. This study was undertaken to define the biological roles of miR-29a in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Methods. Osteoblast-specific miR-29a transgenic mice (Tg) driven by osteocalcin promoter (C57BL/6JNarl-TgOCN-mir29a) or wild-type (WT) mice were given methylprednisolone. Bone mass, trabecular and cortical bone microarchitecture were assessed by μCT. Comparative mRNA and protein expression was quantified by RT-PCR and immunoblotting. Primary bone-marrow mesenchymal cells were isolated for elucidating ex vivo osteogenic and adipogenic differentiation capacity. Results. Decremented miR-29a expression was associated with severe skeletal deterioration and excessive marrow adipogenesis in glucocorticoid-induced osteoporosis bone tissue. Tg mice had high bone mass, spacious trabecular bone and thick cortical bone microstructure. Tg mice also had modest responses to the deleterious actions of glucocorticoid on trabecular microstructure and histomorphological characteristics, mineral acquisition and attenuated marrow fat deposition and osteoclast resorption. Ex vivo, miR-29a overexpression promoted bone-marrow mesenchymal progenitor cells differentiation towards osteogenic cells and away from adipogenic lineage cells. Mechanistically, miR-29a via inhibiting histone deacetylase 4 (HDAC4) actions restored acetylation states of osteogenic regulators Runx2 and β-catenin and decreased osteoclastogenic factor RANKL and adipokine leptin expression in bone microenvironments. Conclusions. Glucocorticoid suppression of miR-29a disintegrates the homeostasis between osteogenic and adipogenic activities, thereby impairs bone formation and skeletal integrity. By suppressing HDAC4, miR-29a stabilizes Runx2 and β-catenin signalling that counteracts the adverse effects of glucocorticoid on bone mass and marrow adiposity. This study unveils the anabolic roles of miR-29a in the progression of glucocorticoid-induced bone loss. Sustained miR-29a action is beneficial for protecting against osteoporosis and excessive marrow adipogenesis. Level of evidence. I

INTRODUCTION. In the treatment of nonunions, and other complications of bone repair, an attractive alternative to bone autografts would be the use of a combination of autologous mesenchymal progenitors cells (MSCs), biomaterials and growth factors. Our goal was to determine the therapeutic potential and contribution to the repair process of different sources of mesenchymal stem cells for the treatment of nonunions. METHODS. The right femur of Sprague-Dawley (SD) rats was stabilized with an aluminum plate (20 mm long, 4 mm wide, 2 mm thick) and four screws (1.5 mm diameter, 8 mm long). A diaphyseal critical size defect was performed (5 mm). Six groups (n=6–8 animals each) were created. A nonunion group (Control group, empty defect); LBA group, live bone allograft; BMP2 group, rhBMP-2 (2 μg) in collagen sponge; PCL group, polycaprolactone scaffold; PMSCs group, PCL scaffold loaded with 5×10. 6. periosteum-derived MSCs; and BMSCs group, PCL scaffold loaded with 5×10. 6. bone marrow-derived MSCs. For cell tracking purposes, LBA and MSCs were derived from SD-GFP transgenic rats. The repair process was followed up by x-rays up to sacrifice, week 10. After sacrifice, femurs were analyzed by micro computed tomography (μCT), histology and immunohistochemistry. For multiple comparisons one-way ANOVA followed by Dunnett”s test for single comparisons was used. Statistical significance was established for p<0.05. RESULTS. Control group did not show healing during follow up or by μCT and histological analysis. Treatment groups BLA and BMP2 showed full healing by week 10 (LBA, 6 out of 6 animals; BMP2, 4 out of 6 animals). The repair callus was quantified by mCT, Control group showed limited formation of bone (11.47±2.01 mm. 3. ) while both LBA and BMP2 groups showed increased bone formation by week 10 when compared with control group (LBA, 35.36±2.24 mm. 3. , p=0.0022; BMP2, 33.32±1.84 mm. 3. , p=0.0022). Histological and μCT analysis confirmed the experimental nonunion model. In PCL treated groups a low number of animals showed radiographic healing: PCL group 1 out of 8 animals; PMSCs group, 2 out of 6 animals; BMSCs group, 0 out of 6 animals. Interestingly, quantification of the repaired callus showed that only PMSCs group produced a significant volume of bone when compared with the Control group (PMSCs, 24.97±6.03 mm. 3. , p=0.0411). PCL and BMSCs groups do not produced significant amount of bone in the repair callus (PCL, 19.00±4.25 mm. 3. , p=0.3095; BMSCs, 12.88±2.38 mm. 3. , p=0.9372). Healing was confirmed by histology and μCT analysis. Finally, the engraftment of transplanted cells was analysed by immunohistochemistry (anti-GFP antibody). Of the three groups receiving cells only the LBA group showed positive signal for GFP at week 10-post surgery. CONCLUSIONS. In conclusion, periosteum-derived progenitor cells are suitable for mimetic autograft design although integration is not yet achieved

Summary Statement. The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures. Introduction. Mesenchymal progenitor cells are capable of generating a wide variety of mature cells that constitute the connective tissue system. Our Laboratory has been developing SMAA GFP reporter mice to prove to be an effective tool for identifying these cells prior to the expression of markers of differentiation characteristic of bone, fat, muscular blood vessels or fibrocartilage. Dkk3 was chosen as a candidate reporter because microarray of SMAA-sorted cells culture indicated high expression of this non-canonical anti-Wnt factor, which was not anticipated in a culture with strong osteogenic potential. Material and Methods. Fracture healing process was evaluated in 12 week old male mice at 3, 5, 7, 14, 21 and 28days post fracture. A 3 color reporter mouse was generated by crossing SMAA-GFPcherry × Col3.6GFPcyan × Dkk3-eGFP and subjected to tibial fracture. A closed transverse fracture was performed by Einhorn device under isoflurane anesthesia after insertion of intramedullary pinning. Longitudinal 5 mm non-calcified cryosections were stabilised with Cryofilm tape. Results. Three days post fracture, the proliferating SMAA-red cells were also beginning to express either Dkk3 or Col3.6. By day 5 the two populations had diverged with the Dkk3 cells being on the outer surface of the developing callus while the Col3.6 cells were forming bone at the base of the callus. By day 7 when the callus is filled with cartilage, Dkk3 is active in cells that are in transition from elongated cells on the external surface of the callus to fibrocartilagenous cells that now express low levels of Col3.6. The zone of cells that express Dkk3 appear to block the passage of the surrounding vasculature into the underlying cartilage and does not deposit fibronectin. By day 14–21 when the cartilage core is resorbed, the only remaining Dkk3 is located in the newly formed periosteum external to the active endocortical bone forming activity associated with the inward remodeling of the outer cortical shell. Discussion. We interpret these findings that Dkk3 marks a non-osteogenic limb of the SMAA progenitor population that within the fracture partitions the osteogenic signals away from the surrounding skeletal muscle and the underlying differentiating fibrocartilage. It is a progenitor to cells that form fibrocartilage in the fracture zone as well as the tenascin C positive cells that populate the fibrous zone of the periosteum, and it resides in the cambial zone of the periosteum. Knowing the biological and molecular function of these cells should lead to a fuller appreciation of the pro- and anti-osteogenic factors that regulate skeletal repair

We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with β-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH)₂D₃, a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and mRNA levels in osteoblast-related genes were enhanced.

Our results suggest that the haemarthrosis induced by injury to the anterior cruciate ligament contains osteoprogenitor cells and is a potential alternative source for cell-based treatment in such injury.

Objectives

This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols.

External fixation of distal tibial fractures is often associated with delayed union. We have investigated whether union can be enhanced by using recombinant bone morphogenetic protein-7 (rhBMP-7).

Osteoinduction with rhBMP-7 and bovine collagen was used in 20 patients with distal tibial fractures which had been treated by external fixation (BMP group). Healing of the fracture was compared with that of 20 matched patients in whom treatment was similar except that rhBMP-7 was not used.

Significantly more fractures had healed by 16 (p = 0.039) and 20 weeks (p = 0.022) in the BMP group compared with the matched group. The mean time to union (p = 0.002), the duration of absence from work (p = 0.018) and the time for which external fixation was required (p = 0.037) were significantly shorter in the BMP group than in the matched group. Secondary intervention due to delayed healing was required in two patients in the BMP group and seven in the matched group.

RhBMP-7 can enhance the union of distal tibial fractures treated by external fixation.

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.