Summary Statement. Demineralised bone matrix augmented tendon-bone fixations in the animal model show less scar tissue and an enthesis morphology closer to the physiologic one which may lead to a more resistant repair construct. Introduction. Rotator cuff repair is one of the most common operative procedures in the shoulder. Yet despite its prevalence recurrent tear rates of up to 94% have been reported in the literature. High failure rates have been associated with tendon detachment from bone at the tendon – bone interface. Exogenous agents as biological strategies to augment tendon – bone healing in the shoulder represent a new area of focus to improve patient outcomes. Demineralised bone matrix (DBM) contains matrix bound proteins, exposed through acid demineralization step of DBM manufacture, and has long been recognised for its osteoinductive and osteoconductive properties. We hypothesised that DBM administered to the bone bed prior to the reattachment of the tendon, will upregulate healing and result in enhanced tissue morphology that more closely resembles that of a normal enthesis. An established ovine transosseous equivalent rotator cuff model was used. Methods. Following ethics approval, 10 adult wethers (18 months) were randomly allocated to control, n=4 (without DBM) or DBM, n=6 (DBM administered to bone bed) groups. The infraspinatus tendon was detached from its insertion and repaired in a transosseous equivalent fashion using PEEK suture anchors. In treatment animals 0.25cc of ovine DBM, previously prepared using a modified Urist protocol, was injected into two drill holes within the bony tendon footprint. Animals were culled at 4 weeks following surgery and processed for tissue histology and microcomputed tomography (μCT) endpoints. Results. No infection or tendon detachment following repair was noted in either group. 3D reconstructed images of μCT scans verified correct DBM and suture anchor placement. Histological images demonstrated distinct differences in tissue morphology between the two groups; however there was no evidence of the four – zoned structure characteristic of a healthy tendon bone insertion, in any specimens. In the control group specimens, the tendon midsubstance was highly disorganised with randomly arranged collagen fibres and diminutive areas of fibrocartilage. In the treatment group, large regions between tendon and bone were occupied by fibrocartilage. Within the fibrocartilage region, insertional collagen fibres appeared organised and chondrocytes were orientated in the direction of the insertional collagen fibres. Organised collagen fibre orientation within the tendon midsubstance was observed, though this was not consistent throughout all the specimens. DBM particles were resorbed and trabecular bone occupied the DBM holes. The PEEK anchors were all in direct contact with the ongrowing bone indicating good quality integration and fixation. Discussion. This study showed that DBM augmented tendon to bone repair leads to an upregulated cellular activity resulting in increased amounts of fibrocartilage between the repaired tendon and underlying bone. The upshot of this is an improved tissue organization which more closely resembles the morphology of the normal enthesis. Introduction of osteoinductive DBM at the tendon – bone interface during surgery may reduce failure rates associated with rotator cuff repair and improve clinical outcomes

Background. Re-attachment of tendon to bone is challenging with surgical repair failing in up to 90% of cases. Poor biological healing is common and characterised by the formation of weak scar tissue. Previous work has demonstrated that decellularised allogenic demineralised bone matrix (DBM) regenerates a physiologic enthesis. Xenografts offer a more cost-effective option but concerns over their immunogenicity have been raised. We hypothesised that augmentation of a healing tendon-bone interface with DBM incorporated with autologous mesenchymal stem cells (MSCs) would result in improved function, and restoration of the native enthesis, with no difference between xenogenic and allogenic scaffolds. Methods. Using an ovine model of tendon-bone retraction the patellar tendon was detached and a complete distal tendon defect measuring 1 cm was created. Suture anchors were used to reattach the shortened tendon and xenogenic DBM + MSCs (n=5) and allogenic DBM + MSCs (n=5) were used to bridge the defect. Functional recovery was assessed every 3 weeks and DBM incorporation into the tendon and its effect on enthesis regeneration was measured using histomorphometry. Results. By 12 weeks, DBM augmentation resulted in significantly improved functional weight bearing with no failures in either group. Compared to xenogenic DBM, allogenic DBM was associated with significantly higher functional weight bearing at 6 (P=0.047), 9 (P=0.028) and 12 weeks (P=0.009). This was accompanied by a more direct type of enthesis characterised by significantly more fibrocartilage and mineralised fibrocartilage. Xenograft was also associated with an immunogenic reaction despite preoperative decellularisation. Conclusion. This study shows that DBM enhances tendon-bone healing and may reduce the high failure rates associated with surgery. An immunogenic reaction, and inferior biomechanical and histological results were also associated with the use of xenograft. Allogenic DBM with autologous MSCs may be a suitable scaffold for the enhancement of tendon-bone healing in the clinical setting. Disclosures. Funded by IKC PoC grant awarded by the University of Leeds. Ethical approval. Granted by the study institution (University College London)

Demineralized bone matrix (DBM) is a natural, collagen-based, well-established osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM osteoinductivity. Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1–2 mm), medium (M, 0.5–1 mm), and small (S, < 0.5 mm). After demineralization, the three DBM samples were characterized by DTA analysis, XRD, ICP-OES, and FTIR. Data clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. The in vivo study showed that only DBM-M was able to induce new bone formation in a subcutaneous ectopic mouse model. When sheep MSC were seeded onto DBM particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. Gene expression analysis performed on recovered implants supports the histological results and underlines the supportive role of MSC in DBM osteoinduction through the regulation of host cells. In conclusion, our results show a relation between DBM particle size, structural modification of the collagen and in vivo osteoinductivity. The medium particles represent a good compromise between no modification (largest particles) and excessive modification (smallest particles) of collagen structure, yielding highest osteoinduction. We believe that these results can guide researchers to use DBM particles of 0.5–1 mm size range in applications aimed at inducing new bone formation, obtaining results more comparable and reliable among different research groups. Furthermore, we suggest to carefully analyze the structure of the collagen when a collagen-based biomaterial is used alone or in association with cells to induce new bone formation

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres

INTRODUCTION

Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development.

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma

Type-2 Diabetic (T2D) patients experience up to a 3-fold increase in bone fracture risk[1]. Paradoxically, T2D-patients have a normal or increased bone mineral density when compared to non-diabetic patients. This implies that T2D has a deleterious effect on bone quality, whereby the intrinsic material properties of the bone matrix are altered. Creating clinical challenges as current diagnostic techniques are unable to accurately predict the fracture probability in T2D-patients. To date, the relationship between cyclic fatigue loading, mechanical properties and microdamage accumulation of T2D-bone tissue has not yet been examined and thus our objective is to investigate this relationship. Ethically approved femoral heads were obtained from patients, with (n=8) and without (n=8) T2D. To obtain the mechanical properties of the sample, one core underwent a monotonic compression test to 10% strain, the other core underwent a cyclic compression test at a normalized stress ratio between 0.0035mm/mm and 0.016mm/mm to a maximum strain of 3%. Microdamage was evaluated by staining the tissue with barium sulfate precipitate [2] and conducting microcomputed tomography scanning with a voxel size of 10μm. The monotonically tested T2D-group showed no statistical difference in mechanical properties to the non-T2D-group, even when normalised against BV/TV. There was also no difference in BV/TV. For the cyclic test, the T2D-group had a significantly higher initial modulus (p<0.01) and final modulus (p<0.05). There was no difference in microdamage accumulation. Previous population-level studies have found that T2D-patients have been shown to have an increased fracture risk when compared to non-T2D-patients. This research indicates that T2D does not impair the mechanical properties of trabecular bone from the femoral heads of T2D-patients, suggesting that other mechanisms may be responsible for the increased fracture risk seen in T2D-patients

Abstract. Objectives. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Methods. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab. Results. Low mRNA expression of PIEZO1 in unloaded cells was upregulated 5-fold following 1-hr of mechanical load (p=0.003). In addition, the transcription factor NFATc1, a known regulator of Piezo1 mechanotransduction, was also upregulated by load (2.4-fold; p=0.03). Y201 cells differentiated in gels expressed the osteocyte marker, SOST. Yoda1 upregulated PIEZO1 (1.7-fold; p=0.057), the early mechanical response gene, cFOS (4-fold; p=0.006), COL1A1 (3.9-fold; p=0.052), and IL-6 expression (7.7-fold; p=0.001). Discussion. This study reveals PIEZO1 as an important mechanosenser in osteocytes. Piezo 1 mediated increases in the bone matrix protein, type I collagen, and IL-6, a cytokine that drives inflammation and bone resorption. This provides a direct link between mechanical activation of Piezo 1, bone remodelling and inflammation, which may contribute to mechanically-induced joint degeneration in osteoarthritis. Mechanistically, we hypothesise this may occur through promoting Ca2+ influx and activation of the NFAT1 signalling pathway

Introduction and Objective. Regeneration of cartilage injuries is greatly limited. Therefore, cartilage injuries are often the starting point for later osteoarthritis. In the past, various bioactive glass (BG) scaffolds have been developed to promote bone healing. Due to the fact that they induce the deposition of hydroxyapatite (HA) -the main component of bone matrix, these BG types are not suitable for chondrogenesis. Hence, a novel BG (Car12N) lacking HA formation, was established. Since BG are generally brittle the combination with polymers is helpful to achieve suitable biomechanic stability. The aim of this interdisciplinary project was to investigate the effects of biodegradable polymer Poly(D,L-lactide-co-glycolide) (PLLA) infiltration into a Car12N scaffold for cartilage tissue engineering. Materials and Methods. BG scaffolds were infiltrated with PLLA using phase separation within a solvent. Pure BG Car12N scaffolds served as control. To assess whether the polymer was homogeneously distributed the polymer to glass ratio and pore contents in the upper, middle and lower third of the scaffolds were examined by light microscopy. For a more precise characterization of the scaffold topology, the glass strut length, the glass strut diameter and the pore circumference were also measured. Leaching tests in 0.1M HCl solution over 8 days were used to allow a gel layer formation on the scaffolds surface. Non-leached and leached scaffolds were subjected to strength testing. Cytotoxicity of the scaffolds with and without polymer was tested according to standards. Scaffolds were colonized with 27.777.8 per cm. 3. primary porcine articular chondrocytes (pACs) or primary human mesenchymal stromal cells (hMSCs), respectively. After cultivation for up to 35 days, the vitality, quantitative DNA and sulfated glycosaminoglycan (sGAG) contents per scaffold were determined. Results. The polymer distribution was not homogeneous in the scaffolds. There were significant differences in glass strut length and pore size. Leaching increased the biomechanical strength. All scaffolds were not cytotoxic. pACs and hMSCs were able to adhere to the scaffold with and without polymer and remained viable during the whole culturing period of 35 d. The DNA content was higher in the pAC colonized scaffolds with polymer than without polymer. The sGAG content was higher in hMSCs seeded scaffolds with polymer than in pACs seeded ones with polymer. Conclusions. Polymer infiltration leads to an increase in mechanical stability of Car12N scaffolds and chondrogenic cells are able to colonize these composites suggesting them as a promising

Background. Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function. Methods. Adult human SSCs were immunoselected, enriched using STRO-1 antibody and cultured on control and test surfaces for 21 days in vitro. The test groups comprised Ti-coated substrates with planar or modified surfaces with nanopillar. Osteoinductive potential was analysed using qPCR and immunostaining to examine gene expression and protein synthesis. Results. Following in vitro (n=5) culture on nanopillars, the expression of osteogenic genes (ALP, Collagen 1, OPN and OCN) and of Osteopontin protein (a bone matrix protein), on Ti pillars were both significantly enhanced when compared to control or Ti planar surfaces. Conclusions. Discrete raised surface nanopillars modulate adult SSC populations in the absence of any chemical cues and enhance their osteogenic properties, an effect not observed on planar Ti constructs. Hence, these findings herald exciting opportunities to improve the implant surface design, implant osseointegration, and, ultimately, implant survival. Level of evidence. Original experimental study

Spinal fusion is one of the most common surgical procedures in spine surgery, whose primary objective is the stabilization of the spine for the treatment of many degenerative, traumatic and oncological diseases of the spine. Autologous bone is still considered the “gold standard” technique for spinal fusion. However, biomaterials which are potentially osteogenic, osteoinductive and osteoconductive can be used to increase the process of spinal fusion. We evaluated two new bone substitutes as an alternative to autologous bone for spinal fusion, using an animal model of large size (adult sheep). A preclinical study was designed to compare the efficacy of SINTlife® Putty and DBSINT® biomaterials with conventional bone autograft in an ovine model of lumbar spine fusion. SINTlife® is a biomaterial made from hydroxyapatite enriched with magnesium ions, resulting to be very similar to natural bone. DBSint® is a paste composite bone, osteo-inductive, pliable and conformable, consisting of demineralized bone matrix (DBM) carried by hydroxyapatite biomimetics. Eighteen adult female sheep were selected for two-levels spine surgical procedures. The animals were divided in two groups: in Group A, one fusion level was treated with SINTlife® Putty and the other level received cortical-cancellous bone autograft; in Group B, one fusion level was treated with DBSINT® and the other level received cortical-cancellous bone autograft. At the end of the experimental time, all the animals were euthanized. The spine segments were analyzed macroscopically, radiographically, microtomographically, histologically and histomorphometrically. The SINT-Life® Putty shows a perfect osteointegration in all the histological specimens. A high percentage of newly formed bone tissue is detected, with lots of trabeculae having structure and morphology similar to the pre-existing bone. In all the specimens collected from DBSINT®-treated animals the presence of hydroxyapatite alone is reported but not the demineralized bone matrix. The presence of newly formed bone tissue can be detected in all the specimens but newly formed bone shows very thin and irregular trabeculae next to the cartilage zone, while away from the border of ossification there are thicker trabeculae similar to the pre-existing bone. The use of the experimental biomaterial SINT-Life® Putty in an ovine model of spine fusion leads to the development of newly formed bone tissue without qualitative and quantitative differences with the one formed with autologous bone. The experimental material DBSINT® seems to lead to less deposition of newly formed bone with wider intertrabecular spaces. Following these results, we planned and submitted to the Ethical Committee a clinical study to evaluate the safety and efficacy of SINT-Life® product in comparison to autologous bone, as an alternative treatment for spine fusion procedures

Osteoporosis is a worldwide spread, silent disease steadily increasing due to demographic shift; it results in bone loss and increased porosity that lead to an increase in bone fragility and to low-energy fractures. In such a contest, we worked on the development of 3D scaffolds engineered to mimic the features of human healthy bone. Healthy and osteoporotic bone microCT scans were obtained from tissues discarded during surgical interventions (Istituto Ortopedico Rizzoli-Italy). The obtained .STL file was used to 3D print a type I collagen solution to mimic bone matrix whereas mesoporous bioactive glass/nano-hydroxyapatite were embedded within the collagen fibers to mimic the inorganic phase of human bone. The rheological properties of the Type I collagen/mesoporous glass suspensions were investigated at different collagen concentration and temperatures. The possibility of incorporating growth factors (IGF and β-TGF) in the scaffold struts was investigated proposing several approaches and their retained activity was assessed. Different co-culture of osteoblasts and osteoclasts set-ups were explored in order to define the influence of both chemical and topographical stimuli on the osteoblast-osteoclast coupling

One of the latest trends in the field of tissue engineering is the development of in vitro 3D systems mimicking the target tissue or organ and thus recapitulating the tridimensional structure and microenvironment experienced by cells in vivo. Interestingly, certain tissues are known to be regulated by endogenous bioelectrical cues, in addition to chemical and mechanical cues. One such tissue is the bone. It has, indeed, been demonstrated to exhibit piezoelectric properties in vivo, with electrical signaling playing a role in its formation during the early embryo developmental stages. Electrical stimulation has been proven to sustain cell proliferation and to boost the expression of relevant genes and induce higher levels of enzymatic activities related to bone matrix deposition. Herein, we describe the development of a 3D model of bone tissue based on the conductive polymer PEDOT:PSS and human adipose derived stem cells. 3D electroactive porous scaffolds have been produced using the ice-templating technique, and different compositions (different ratios of conductive polymer to Collagen Type 1) have been explored. The developed scaffolds as well as cells interaction and response have been characterized. Overall, the results obtained so far highlight the usefulness of the porous conductive scaffolds as an in vitro platform for the development of 3D models for bone tissue engineering

During remodelling, osteoclasts produce discrete bone cavities filled with bone and this is associated with the dimensions of the cavity. The aim of this study is to investigate the effect of pores of similar size to those produced by osteoclasts on the morphology, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. The hypothesis is that a porous surface similar in morphology to a bone surface prepared by osteoclasts will increase cell proliferation and osteogenic differentiation of MSCs. Sheep BMSCs were seeded onto plain titanium surfaces and 100µm, 250µm and 500µm discrete pores surfaces. Cell metabolic activity was investigated using Presto Blue on days 3, 7 and 10. Bone mineralisation was quantified by Alizarin red staining at days 3, 7 and 14. Cell morphology was observed by scanning electron microscopy (SEM). Data was statistically analysed using one-way analysis of variance and a Bonferroni correction method. Cells on porous discs had a three dimensional phenotype and aligned on the circumference of each pore. Metabolic activity was significantly higher by day 10 on plain discs compared to all porous discs. Bone mineralization was significantly higher on 100µm pores by day 3 (0.545mM±0.66; p=0.047) than plain discs and significantly higher on both 100µm and 250µm pores by day 7(p=0.000 and p=0.005) than plain discs. Substantial mineralised bone matrix was found on 100µm discs without being treated with osteogenic supplements, compared to other control disc types (p=0.043, p=0.003, p=0.000). The different topographies altered cell behaviour and migration.100µm pores demonstrated earlier and enhanced bone mineralisation even in the absence of osteogenic supplements. This pore size is aligned to the size of individual resorption bays that osteoclasts produce on bone surfaces and is considerably lower than the pore sizes used to enhance osteo-integration of implant surfaces

Skeletal sequels of traumatisms, diseases or surgery often lead to bone defects that fail to self-repair. Although the gold standard for bone reconstruction remains the autologous bone graft (ABG), it however exhibits some drawbacks and bone substitutes developed to replace ABG are still far for having its bone regeneration capacity. Herein, we aim to assess a new injectable allogeneic bone substitute (AlloBS) for bone reconstruction. Decellularized and viro-inactivated human femoral heads were crushed then sifted to obtain cortico-spongious powders (CSP). CSP were then partly demineralized and heated, resulting in AlloBS composed of particles consisting in a mineralized core surrounded by demineralized bone matrix, engulfed in a collagen I gelatin. Calvarial defects (5mm in diameter, n=6/condition) in syngeneic Lewis1A rats were filled with CSP, AlloBS±TBM (total bone marrow), BCP (biphasic calcium phosphate)±TBM or left unfilled (control). After 7 weeks, the mineral volume/total volume (MV/TV) ratios were measured by µCT and Movat's pentachrome staining were performed on undemineralized frontal sections. The MV/TV ratios in defects filled with CSP, AlloBS or BCP were equivalent, whereas the MV/TV ratio was higher in AlloBS+TBM compared to CSP, AlloBS or BCP (p<0.01; Mann-Whitney). Histological analyses exhibited a collagen-rich matrix in all the defects, and osteoid at the surface of all implanted biomaterials. Our data indicates that AlloBS is a promising candidate for bone reconstruction, with ease of manipulation, injectability and substantial osteogenic capacity. Further experiments in larger animal models are under consideration to assess whether AlloBS may be a relevant clinical alternative to ABG

Osteophyte deposition and subchondral bone damage are notable features of osteoarthritis (OA). Deregulated mineralization contributes to osteophyte and subchondral irregularity. The microRNA-29 (miR-29) family is associated with arthritic disorders. This study is aimed to investigate miR-29a function to OA osteophyte formation and subchondral integrity. Intact and damaged articular cartilage in patients with end-stage knee OA who required total knee arthroplasty were harvested to probe miR-29a, cartilage, and mineralized matrix expression using RT-PCR and in situ hybridization. Osteophyte volume and subchondral morphometry of collagenase-induced OA knees in mice were quantified using μCT and histomorphometry. Increased bone matrix expression (collagen I and bone alkaline phosphatase) and reduced cartilage matrix (collagen II and aggrecan) along with low miR-29a expression existed in human OA specimens. Aged miR-29a knockout mice showed spontaneous osteophyte formation and articular cartilage erosion. In primary articular chondrocytes, miR-29a deficiency significantly reduced cartilage matrix synthesis, whereas von Kossa staining-positive mineralized matrix production was increased. Of interest, the severity of collagenase-induced osteophyte accumulation and subchondral damage along with serum cartilage breakdown products CTX-II and COMP levels were significantly compromised in mice overexpressing miR-29a. Intra-articularly injecting miR-29a significantly reduced osteophyte volume and subchondral integrity and retained cartilage morphology in collagenase-injured knees. Reduced miR-29a signalling worsens osteophyte and subchondral destruction in OA through increasing mineralized matrix formation of chondrocytes. Restoring miR-29a shields joints from cartilage degradation, osteophyte and subchondral destruction. This study conveys new mechanistic underlying OA osteophyte pathogenesis and shines light on the remedial potential of miR-29a to OA

Introduction. Amino acids like arginine and lysine have been suggested to hasten the process of fracture healing by improving the local blood supply, supplementing growth factors, and improving collagen synthesis. We studied the role of lysine and arginine in the fracture repair process with regard to the rate of healing, probable mechanisms involved in the process, and mutual synergism between these agents. Materials and methods. In an experimental study, 40 rabbits were subjected to ulnar osteotomy. They were distributed in control (14) and test groups (26). Twenty-six animals in the test group were fed with a diet rich in lysine and arginine. Both the groups were followed radiologically and histologically till union. Results. Ten weeks postoperatively, there was difference evident radiologically between those supplemented with lysine and arginine, indicating that these components enhance the healing in the later part of bone remodeling, canal restoration, and medullary as well as cortical continuity and repair. X-rays obtained at weeks 9, 10, and 12 in both the groups showed statistical significance. These findings showed that healing is better in the test group in terms of increased vascularity in the early part of healing, i.e., at approx. 2–3 weeks and in terms of bone matrix, Haversian system formation, and cortical repair in the later part of healing, i.e., at approx. 9–12 weeks between the two groups. There was better healing of osteotomy in terms of better vascularization, callus formation, and mineralization in the test group. The time of healing in the test group was reduced by a period of 2 weeks. Discussion. NO is expressed during fracture healing in rats and humans, as after fracture, mRNA, protein, and enzymatic activity iNOS have been identified at the fracture callus with maximum activity at day 15. Thus, the initial better healing, by 3 weeks, in the test group rabbits can be explained by the fact that the iNOS activity mediates an increased vascularity at the fracture site. The mRNA activity for eNOS and bNOS was induced slightly later than that for iNOS, which was consistent with a temporal increase in the calcium-dependent NOS activity that gradually increased up to day 30. All calcium-dependent processes like collagen recruitment for Haversian system formation, better bone matrix, and cortical repair were significantly better at any point of time, in the rabbits that were supplemented with arginine; however, lysine has also an important role in these processes. Arginine may influence bone formation by enhancing local IGF-I production. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Arginine being nitric oxide donor increases angiogenesis. Summary. Amino acids like arginine and lysine may hasten fracture healing. Adjuvant amino acid treatment is having inherent advantage in being nontoxic, inexpensive, and a simple oral therapy

The treatment of chronic osteomyelitis often includes surgical debridement and filling the resultant void with antibiotic-loaded polymethylmethacrylate cement, bone grafts or bone substitutes. Recently, the use of bioactive glass to treat bone defects in infections has been reported in a limited series of patients. However, no direct comparison between this biomaterial and antibiotic-loaded bone substitute has been performed. . In this retrospective study, we compared the safety and efficacy of surgical debridement and local application of the bioactive glass S53P4 in a series of 27 patients affected by chronic osteomyelitis of the long bones (Group A) with two other series, treated respectively with an antibiotic-loaded hydroxyapatite and calcium sulphate compound (Group B; n = 27) or a mixture of tricalcium phosphate and an antibiotic-loaded demineralised bone matrix (Group C; n = 22). Systemic antibiotics were also used in all groups. After comparable periods of follow-up, the control of infection was similar in the three groups. In particular, 25 out of 27 (92.6%) patients of Group A, 24 out of 27 (88.9%) in Group B and 19 out of 22 (86.3%) in Group C showed no infection recurrence at means of 21.8 (12 to 36), 22.1 (12 to 36) and 21.5 (12 to 36) months follow-up, respectively, while Group A showed a reduced wound complication rate. Our results show that patients treated with a bioactive glass without local antibiotics achieved similar eradication of infection and less drainage than those treated with two different antibiotic-loaded calcium-based bone substitutes. Cite this article: Bone Joint J 2014; 96-B:845–50

Amniotic membrane (AM) and amnion/chorion foetal membranes (ACM) are mainly composed of collagen & laminin layers and constitute relatively new materials to the dental market. They have proven effective for periodontal treatments such as Guided Tissue Regeneration (GTR) [1–3]. Based on our expertise in the field of lyophilisation & securisation of human bone allograft (Phoenix® process), we aimed to develop our own process applied to ACM and to control its in vivoefficacy in GTR indication. Human placentas were donated under informed consent. ACM were separated from placenta and processed with a proprietary AMTRIX (TBF) Process. Resulting product was called ACMTRIX. The effectiveness of ACMTRIX in GTR was evaluated using an in vivorat calvaria defect model as followed:. Empty defect (2 animals),. ACMTRIX apposed onto the defect (4 animals),. 3 Bone substitutes (allogenic – mineralized cortical bone powder (Phoenix®); demineralized cancellous bone powder mixed with hydroxyapatite and demineralized bone matrix (DBM) cancellous block) filled in the defect and covered by ACMTRIX (4 animals). One animal per study group was sacrificed after 8 weeks, all others after 8 weeks. Evaluations were performed by: macroscopic observations, X Ray micro-CT, and histological analysis. For all groups using ACMTRIX, no major sign of inflammation were observed macroscopically and histologically. Moreover, bone tissue was already mature from 8 weeks and bone filling was slight to moderate. The higher mean rate of mineralization was obtained for the group associating DBM cancellous block + ACMTRIX. Although a xenogenic material, ACMTRIX was very well integrated without significant inflammatory reaction compared to empty defect and fully integrated in subcutaneous area. The mineralization was superior with DBM cancellous block probably thanks to the stabilization of the material in the defect. Used alone, ACMTRIX has no osteogenic potential. In conclusion, ACMTRIX has the potential to function as barrier for GTR and the unique properties associated with this material can augment its potential as a matrix for periodontal regeneration