Introduction. Recently, some case reports have been published, in which nonunions were successfully healed with parathyroid hormone 1–34 (PTH) administration. Previously, we demonstrated that the intervening tissue at the nonunion site contains multilineage mesenchymal progenitor cells and plays an important role during the healing process of nonunion. We investigated the effect of PTH on osteogenic differentiation of human nonunion tissue-derived cells (NCs) in vitro. Hypothesis. We hypothesized that PTH directly promoted osteogenic differentiation of NCs. Materials & Methods. NCs were isolated from 4 patients, and cultured. The cells were divided into two groups: (1) PTH (−) group: cells cultured in osteogenic medium (OM), (2) PTH (+) group: cells cultured in OM with PTH. Osteogenic differentiation potential was analyzed. Results. Real-time PCR analysis showed that gene expression levels of Runx2, ALP, OC and PTHR1 in PTH (+) group were lower than PTH (−) group at day 14. In both groups, there was no significant difference in ALP activity at days 8 and 14, and in the intensity of Alizarin red S staining at day 20. Discussion. Treatment of PTH did not lead to increase osteogenic differentiation of NCs. Nonunion healing by PTH administration may be caused by other mechanisms such as mobilization and recruitment of osteoprogenitor cells

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. The need for a novel, cost effective treatment option for osteochondral defects has therefore never been greater. As an emerging technology, three-dimensional (3D) bioprinting has the capacity to deposit cells, extracellular matrices and other biological materials in user-defined patterns to build complex tissue constructs from the “bottom up”. Through use of extrusion bioprinting and fused deposition modelling (FDM) 3D printing, porous 3D scaffolds were successfully created in this study from hydrogels and synthetic polymers. Mesenchymal stem cells (MSCs) seeded onto polycaprolactone scaffolds with defined pore sizes and porosity maintained viability over a 7-day period, with addition of alginate hydrogel and scaffold surface treatment with NaOH increasing cell adhesion and viability. MSC-laden alginate constructs produced via extrusion bioprinting also maintained structural integrity and cell viability over 7 days in vitro culture. Growth within osteogenic media resulted in successful osteogenic differentiation of MSCs within scaffolds compared to controls (p<0.001). MSC spheroids were also successfully created and bioprinted within a novel, supramolecular hydrogel with tunable stiffness. In conclusion, 3D constructs capable of supporting osteogenic differentiation of MSCs were biofabricated via FDM and extrusion bioprinting. Future work will look to increase osteochondral construct size and complexity, whilst maintaining cell viability

Introduction. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance healing of fracture and nonunion. Bone morphogenetic protein-7 (BMP-7) has also been reported to promote bone formation. Recently, we demonstrated progenitor cells with osteogenic/chondrogenic differentiation potential existed in human fracture hematoma and nonunion tissue. Hypothesis. We hypothesised the combined application of LIPUS and BMP-7 would cause major effect on osteogenesis of hematoma-derived cells (HCs) and nonunion tissue-derived cells (NCs). Materials & Methods. HCs and NCs were isolated, and cultured. The cells were divided into two groups: (1) BMP-7 group: cells cultured in osteogenic medium (OM), and (2) BMP-7 + LIPUS group: cells cultured in OM with LIPUS treatment. LIPUS (30 mW/cm2, intensity at 1.5 MHz) was given for 20 minutes daily. Osteogenic differentiation potential and proliferation were analysed. Results. ALP activity, the gene expression of osteogenic genes, and mineralisation of HCs and NCs were shown to be higher in BMP-7 + LIPUS group than in BMP-7 group. There was no significant difference in cell proliferation between the two groups. Discussion. Our findings demonstrated the significant effect of LIPUS on the osteogenic differentiation of HCs and NCs induced by BMP-7. This study may provide significant evidence for the clinical combined application of BMP-7 and LIPUS for the treatment of severe bone fracture and nonunion

Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Introduction. The concept of “bone graft expanders” has been popularised to increase the volume and biological activity of the implanted Material. HYPOTHESIS. Orthoss® granules support exogenously seeded MSCs and attract neighbouring host MSCs. Methods. In 3-D cultures’ Orthoss® granules were seeded with 2×10. 5. bone marrow MSCs/granule and maintained in MSC expansion or differentiation media for 21 days. In homing experiments’ bone autografts were placed in close proximity to Orthoss®. Scaffold colonisation and MSC differentiation were assessed by confocal microscopy’ standard electron microscopy’ and energy-dispersive X-ray spectroscopy. Results. Long-term incubation of MSC/scaffold resulted in formation of multiple cell-matrix layers lining the scaffold pores as well as outer surfaces. MSC differentiation to osteoblasts was evident as strong deposition of Calcium and Phosphorus was detected in both MSC expansion and osteogenic conditions. Cell egress experiments demonstrated the migration of cells from neighbouring autografts and their attachment and re-settlement on Orthoss®. Discussion & Conclusions. Orthoss® scaffolds support MSC attachment’ growth and osteogenic differentiation whereas resident bone subpopulations can rapidly migrate towards’ attach’ and expand on them. These results indicate that Orthoss® can serve as a graft expander for repairing large bone defects in trauma patients

Introduction. Failures in fracture healing are mainly caused by a lack of neovascularization. We have previously demonstrated that G-CSF-mobilized peripheral blood (GM-PB) CD34+ cells, an endothelial progenitor enriched cell population, contributed to fracture healing via vasculogenesis and osteogenesis. We postulated the hypothesis that local transplantation of culture expanded bone marrow (cEx-BM) CD34+ cells could exhibit therapeutic potential for fracture healing. Materials. BM CD34+ cells were cultured in specific medium with 5 growth factors for 1week. A reproducible model of femoral fracture was created in nude rats with periosteum cauterization, which leads to nonunion at 8 weeks post-fracture. Rats received local administration of the following cells or PBS alone(1)cEx-BM, (2)BM, (3)GM-PB CD34+ cells or (4)PBS. Results. Our 7-day culture expansion technique allowed us to obtain 23 times of BM CD34+ cells maintaining 60% purity of CD34 positivity. cEx-BM CD34+ cells exhibited striking therapeutic efficacy for unhealing fracture promoting neovascularization and osteogenesis in sites of fracture. Moreover, cEx-BM CD34+ cells showed high capacity of colony formation and osteogenic differentiation. Conclusion. BM CD34+ cells can be obtained from the fracture site at the time of primary operation and stored for further use, autologous culture expanded BM CD34+ cell transplantation therapy would be not only a simple but also powerful therapeutic strategy for unhealing fracture

Introduction. iPSCs represent a promising cell source for bone regeneration. To generate osteoprogenitor cells, most protocols use the generation of embryoid bodies (EBs). However, these protocols give rise to heterogeneous population of different cell lineage. Hypothesis. We hypothesized that a direct plating method without EB formation step could be an efficient protocol for generating a homogeneous population of osteoprogenitor cells from iPSCs. Materials & Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. DPCs were cultured for 14 days in osteogenic medium. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity, real-time PCR, and alizarin red S staining. Results. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs. Flow cytometry analysis revealed DPCs had similar characteristics to MSC, suggesting DPCs lost pluripotency. Moreover, the DPCs exhibited high osteogenic potential. Discussion & Conclusion. Our novel direct plating method in the absence of EB formation step could be amenable to large-scale production of osteoprogenitor cells for bone regeneration

Objectives

The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics.

We hypothesised that cells obtained via a Reamer–Irrigator–Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction.

Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

Cite this article: Bone Joint J 2013;95-B:1269–74.

Heterotopic ossification (HO) is perhaps the single most significant obstacle to independence, functional mobility, and return to duty for combat-injured veterans of Operation Enduring Freedom and Operation Iraqi Freedom. Recent research into the cause(s) of HO has been driven by a markedly higher prevalence seen in these wounded warriors than encountered in previous wars or following civilian trauma. To that end, research in both civilian and military laboratories continues to shed light onto the complex mechanisms behind HO formation, including systemic and wound specific factors, cell lineage, and neurogenic inflammation. Of particular interest, non-invasive in vivo testing using Raman spectroscopy may become a feasible modality for early detection, and a wound-specific model designed to detect the early gene transcript signatures associated with HO is being tested. Through a combined effort, the goals of early detection, risk stratification, and development of novel systemic and local prophylaxis may soon be attainable.