Introduction: Bone morphogenetic protein (BMP) has become widely used in the interbody space as part of a lumbar fusion. Complications can occur but are not completely understood or well documented. Methods: A prospective review of consecutive lumbar interbody fusions performed by a single surgeon was undertaken over a 2-year period. Early complications (defined as occurring within the first six weeks) were noted. The interbody cages (titanium Syncage, Synthes cages for ALIFs and PEEK Capstone, Medtronic cages for TLIFs) were filled with Infuse BMP-collagen sponge. Until early 2007, the amount of Infuse used was not strictly measured but after that time, only enough to fill the cage was used, with the volume assessed according to the manufacturer’s guidelines. Patients were routinely assessed preoperatively and at the six-week postoperative review using a visual analogue scale and the Oswestry disability score. Plain x-ray and MRI were obtained preoperatively, and plain x-ray was obtained postoperatively. In addition, if early problems developed, MRI scan was obtained. The incidence of complications was compared to that seen in similar procedures but where BMP was not used. Results: 114 patients, including 78 transforaminal inter-body fusions (TLIFs) and 36 anterior lumbar interbody fusions (ALIFs) were available for review. Early complications were noted in 10 of 114 patients. Two (both with TLIF) were not directly linked with BMP use: in the first, the cage migrated posteriorly and in the second, a deep infection developed. The remaining eight were associated with an exaggerated inflammatory response likely related to BMP use. Severe back pain associated with marked vertebral body inflammation seen on MRI was noted in two ALIF patients. The response occurred within 2 weeks of surgery, and settled with conservative treatment. Severe back pain and recurrence of leg pain developed in six TLIF patients. Fluid cyst formation within the spinal canal was seen on MRI in 4 of these. The cyst extended from the region of the posterior aspect of the cage into the canal and toward the area of the excised facet joint, resulting in compression of the exiting nerve root. In one case, the surgical site was re-explored and the cyst removed. In two cases, the cyst was aspirated under CT guidance and injected with steroid. In the final case a course of oral Prednisone was administered. In the remaining two TLIF cases, there was a diffuse inflammatory response in the region of the posterior aspect of the cage and adjacent epidural space but without discrete cyst formation. In one, oral Prednisone was prescribed. The second was treated expectantly. The majority of these complications were noted in 2007, after the dose of BMP was titrated in line with the manufacturer’s guidelines. In contrast, no such complications were seen when a similar technique but without BMP was undertaken in 33 posterior and 41 anterior interbody fusions. Discussion: The incidence of an exaggerated inflammatory response with BMP in the lumbar spine may be under-recognised. The majority of complications published to date relate to vertebral osteolysis and bony overgrowth, although a number of adverse responses to BMP reported to the FDA relate to fluid cyst formation or inflammation. With the rapid increase in BMP use, it is important that surgeons are aware of potential complications, and possible strategies to prevent and address them

Introduction. In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions. Methods. HACs from five OA patients (passage 1) were cultured in cytokine-free medium, under 280 or 380 mOsm respectively, under standard 2D in vitro conditions. TGF-β2 knockdown (KD) by siRNA was performed in the presence or absence of the established bone morphogenetic protein (BMP) type I receptor (BMPRI) inhibitor dorsomorphin (10 μM). Expression of COL2A1 was evaluated by qRT-PCR. Results. Culturing HACs at 380 mOsm increased COL2A1 mRNA expression. Addition of dorsomorphin decreased COL2A1 mRNA expression at both 280 and 380 mOsm, but its expression was still significantly higher at 380 mOsm. In hyperosmotic 380 mOsm culture conditions, TGF-β2 KD further increased COL2A1 mRNA expression, while addition of dorsomorphin under these conditions abrogated this effect. Still, expression of COL2A1 mRNA levels remained higher as compared to 280 mOsm. Conclusion. This study confirms that BMP signalling is involved in the expression of the single best accepted key chondrocyte marker, COL2A1, in osteoarthritic HACs. However, inhibition of BMP signalling could not abrogate the increase in COL2A1 expression under hyperosmotic culture conditions. Our data suggest an inverse regulation of TGF-β2 and COL2A1, under these conditions, which may largely be dependent on increased BMPRI-mediated cell signaling. Our findings further suggest that hyperosmotic culture improves COL2A1 expression by means that are independent of TGF-β- and BMPRI-signaling. Further elucidation of the molecular network underlying this observation is ongoing

Numerous investigators have described osteogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage. Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads

As cartilage has poor intrinsic repair capacity, in vitroexpansion of human articular chondrocytes (HACs) is required for cell-based therapies to treat cartilage pathologies. During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. It has been shown that physiological osmolarity (i.e. 380 mOsm) increases collagen type II (COL2) expression in vitro, but the underlying molecular mechanism is unknown. Transforming growth factor beta (TGFβ) super family members are accepted key regulators of chondrocyte differentiation and known to stimulate COL2 production. In this study we aimed to elucidate the role of TGFβ superfamily member signalling as a molecular mechanism potentially driving the COL2 expression under physiological (380 mOsm) culture conditions. HACs from OA patients (p1) were cultured in cytokine-free medium of 280 or 380 mOsm, under standard 2D in vitroconditions, with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, BMPs and chondrocyte marker genes was evaluated by QPCR. TGFβ2 protein secretion was evaluated using ELISA and bioactivity was determined using an established reporter cell line. Involvement of BMP signaling was investigated by culturing OA HACs (p1) in the presence or absence of dorsomorphin (10 µM). Physiological osmolarity increased TGFβ2 and TGFβ3 mRNA expression, TGFβ2 protein secretion as well as general TGFβ activity by 380 mOsm. Upon TGFβ2 isoform-specific knockdown COL2 mRNA expression was induced. TGFβ2 RNAi induced expression of several BMPs (e.g. BMP2,-4,-6) and this induction was enhanced in culture conditions with physiological osmolarity. Dorsomorphin inhibited physiological osmolarity induced COL2 mRNA expression. TGFβ2 knockdown under 380 mOsm increases COL2 expression in human osteoarthritic chondrocytes in vitromost likely through a regulatory feedback loop via BMP signaling, which is involved in osmolarity-induced COL2 expression. Future studies will further elucidate the BMP-mediated regulatory feedback loop after TGF β2 knockdown and its influence on COL2 expression

Numerous investigators have described chondrogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications. Therefore, the primary goal of this research endeavor is to achieve chondrogenic differentiation of ADSC. Previous work had also demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for postnatal maintenance of articular cartilage. In fact, cartilage within the joints of transgenic mice deficient in BMP receptor 1A rapidly degenerates after birth in a process resembling accelerated human osteoarthritis. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our isolated stem cells in order to direct their differentiation into the chondrocyte lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized to screen and enrich the ADSC population for stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve chondrogenic differentiation after 13 to 16 days of in vitro culture, as revealed by immunohistochemistry assays for the bio-markers of articular cartilage (type II collagen and the proteoglycan aggrecan). Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous chondrocytes in vitro for future implantation at the site of osteochondral defects. This method of attaining cartilaginous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance during chondrogenesis of signaling mediated by the BMP receptor 1A. Further tissue engineering studies are now in progress to evaluate the ability of ADSC to differentiate into chondrocytes after seeding onto poly-caprolactone polymer scaffolds

Healing of tendons is sensitive to mechanical loading, and the callus strength is reduced by ¾ after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This suggests that BMP signalling is crucial during tendon healing, and that it is influenced by mechanical loading. We investigated if mechanical loading influences BMP signalling in intact and healing tendons, and how BMP gene expression changes during healing. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. Gene expression for OP-1, GDF-5, -6, -7, Follistatin, Noggin, BMP-receptor 1b and BMP-receptor 2 were analysed with real-time PCR. Loading had no detectable effects on intact tendons. During repair, loading decreased follistatin by more than half (p=0.0001), and increased GDF-5 (p=0.02). All genes showed changes during repair (p=0.0001), but the time sequences differed. GDF-5 and GDF-7 were generally more expressed than OP-1 and GDF-6. GDF-5 and GDF-7 were more expressed in normal tendons than during repair. Noggin was never detected. Our results suggest that GDF-5 is specific for the mature tendon, and not much involved in repair. This contrasts to GDF-7, which is involved in both. OP-1 and GDF-6 seem to be involved in early healing. There was less expression of follistatin in loaded tendons during healing. The mechanosensitivity is likely of most importance at day 14 and 21 since the difference in strength between loaded and unloaded tendons is huge. An Anova with only these time points reveals effects of loading on GDF-5 and follistatin (p=0.0001 for both) and significant differences between the days for most variables

Purpose of the study: Drilling along yields disappointing results for osteonecrosis of the femoral head due to the high failure rate despite prolonged rest and also because of the risk of fracture. To prevent these problems, we have developed a new drilling technique which was evaluated prospectively. Material and methods: The procedure performed percutaneously uses a lateral cortical orifice measuring 5mm, non-concentrated autologous bone marrow was injected after drilling. Osteoinductive protein (BMP7) was associated in random fashion (groups BMP+ and BMP−). Ficat stage 1 and 2 necrosis was included. Outcome was the rate or revision for prosthesis. Results: Forty hips (36 patients) were included and assessed at mean four years (range 2–6). The necrosis was related to: alcoholism (n=5), cortisone (n=25), barotraumas (n=2), metabolic disease (n=4), idiopathic condition (n=4). Group BMP- (drilling+bone marrow) included 24 hips and group BMP+ (drilling+bone marrow+BMP7) 16 hips. The groups were comparable regarding necrosis stage (15% stage 3, 65% stage 2, 20% stage 1) and mean Koo index (27 BMP+ vs 34 BMP-; NS). There were no infections and no fractures despite immediate and complete weight-bearing. The revision rate for prosthesis was higher in the BMP- group (67%) than in the BMP+ group (43%) but the difference did not reach significance (p=0.10). The failure rate was not affected by the severity of the necrosis in the BMP+ group: all stage 3 hips were revised in the BMP- group versus none in the BMP+ group. The only variable predictive of revision for prosthesis was the Koo index (p=0.02). Discussion: Adjunction of BMP did not improve significantly the success rate of drilling with bone marrow adjunction but adding BMP appeared to limit the unfavourable impact of server necrosis observed in the BMP- group. To reach a statistical power of 80%, 40 cases would be needed in each arm. This threshold has not yet been reached. It can be noted however that the proposed method does ensures early weight bearing without the risk of complications. Similarly, since it is a percutaneous procedure, later arthroplasty is not compromised. The principle confounding factor, the richness of the bone marrow, was not assessed, motivating a new randomized trial with measurement of CFU-F

Introduction. We describe a minimally invasive technique that permits intra-focal bone graft of non-union sites with minimal disturbance of soft tissues and vascularity, and present the results of this technique. Materials and Methods. 10 patients with established tibia fracture non-union were judged suitable for the technique, and were treated in our limb reconstruction unit between January 1995 to June 2007. Eight patients were male, 2 were females with a mean age of 37.4 years (27–64). Five fractures were in the distal tibia and five were diaphyseal fractures. Five fractures were as a result of high velocity and 6 fractures were open. Average number of previous operations were 3 (range 1–7). Time lapse between injury to trephine grafting procedure was mean of 34 (6–168 months). 5 patients had a sedentary job, 2 were labourers and 2 were not working. There were 5 smokers and 2 obese patients. Six cases were of infected non-unions. Operative technique. Under general anaesthesia, the graft(s) was first obtained from the iliac crest using a guide wire centred trephine. A stab incision was made at the non-union site under image control. A core was taken across the non-union, taking care to breach both bone fragments. A trephine core was rolled in Bone Morphogenetic Protein (BMP-7). This was then introduced through the trephine in to the non-union site and pushed with the plunger into the defect created by the cylindrical plug taken from the site. After this, the trocar with the stylet in place was gradually withdrawn with small oscillating motions to fill in the path of the trocar. This was repeated about 3–4 times at the non-union site. The stab wound were closed with interrupted Nylon. Results. 10 patients underwent treatment for complex non-union after initial operation (average number 3) at the referring centre. Treatment in a circular fixator ranged from 10–33 months (20.7 months) with reapplication in 2 patients. Average number of procedures were 4 (range 1–7). Trephine grafting was performed in all cases; BMP was used in 4 cases along with iliac crest bone graft. All patients attained clinical and radiological union with CT confirmation required in 2 patients. 4 patients with sedentary job returned to work, manual workers returned to low demand occupation. Conclusion. The technique described is a safe and effective treatment method for this complex problem with limited donor site morbidity and avoids prolonged hospital stay

Introduction: Humerus non union is unfrequent, and reported series short. New fixation with or without autograft remain the gold standard to achieve bone union in 95% of cases. But no report are published in case of failure of that new procedure. 9 patients with a failure of autograft in humerus non union have been treated by new fixation an adjonction of BMP. Matériel et méthodes: 9 patients with an average age of 53,8 yo (24–71) have been treated and followed prospectively for a minimum time of 3 years. The delay between the fracture and the secon procedure was 31 months (6–103). The number of procedure after the fracture fixation was 1,4 (1–5). In 6/9 cases a technical pitfall during the initial procedure was pointed. In 3/9 cases a radial palsy associated with the initial fracture, a septic condition of the non union, general risk factors of non union (diabetes, tabac) and a non collaborative patient were reported. Bone union was defined as the continuity of 4/4 cortex on Xray (AP and sagital plane) and or with ct scan. Osigraft. ®. (BMP7) was implanted in the resected zone of non union which was fixed with 2 plates after reaming and decortication. Résults: No complication have been reported. One case failed (septic non union, 3 procedures, very active patient). The 8 last patients achieved bone union with a delay of 11,1 mois (6–14) without any additive procedure. The 3 septic cases have been solved. Shoulder and elbow function were good without nerves complications. Discussion:: Autograft remains the gold standard in term of treatment of non union. But nothing is reported in humerus non union if iliac crest autograft have failed to achieve non union. In such an indication (failure after an autograft) and in such a level (humerus can be shorten) a stable fixation an a growth factor allowed to solve resistant cases of non union even in septic conditions. The failure of the initial treatment of the fracture (unstable fixation, unfilled bone’s defect) remain the main cause of non union

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms.

Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2^-/-). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR.

Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones.

Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.

We present a retrospective study of comparision between two types of aritifical boen graft substitues. There is an overwhelming marketting drive on part of companies to sell alternative bone grafts/BMP. We in this study compae two such producsts and their cost effectiveness. This is an interventional, retrospective, non consecutive, non randamised case series study of 27 patients. Type I bone graft is Mini MIIG which is surgical grade calciun sulphate which is osteoconductive. Type II bone graft is Allomatrix which conatins bone marrow aspirate, bone morphogenic protein, concellous bone chips and surgical grade calciun sulphate which is osteogenic, osteoinductive and osteoconductive. In this study 14 cases were treated with Mini MIIG and 18 with Allomatrix. There were 24 primary fractures with bone defect, 2 non union and 1 delayed union. Complete bony union were seen in all 27 patients. Average time to heal since bone grafting is 3 months. Complications are extrubent callus formation, bone formatiom in soft tissue, but no patient required secondary procedure to trim the bone. Cost for Allomatrix is £ 356.00 and Mini MIIG is £348.00. Use of such artificial bone grafting avoids the complication of autografting which includes bone graft side morbidity like pain, bleeding and neurvascular damage. For fresh fractures useage of such artificial bone grafts doesnt shorten the healing time, doesnt prevent collapse at fracture site and it is not cost effective. For non union and delyaed unions it avoids the cost for artifical bone grafting. But autograft also incurs the cost of removing, theatre timing. human resources cost and hospital inpatient costs. There is no difference between one type of bone graft over the other and for fresh fracture both of them has no advantage over using no bone grafts. Our study concludes artifical bone graft is of no advantage for fresh fractures and for non union and delayed unions it is too small a number to come to any conclusion

Introduction

Osteoarthritis (OA) is a slow progressive disease and a huge economic burden. A new target for therapy could be a growth factor treatment to prevent the loss of cartilage following injuries to the joint. BMP-7 is a promising candidate for such a novel therapy based on growth factors. In this study we combined the chondroprotective effects of BMP-7 with a novel thermosensitive hydrogel to prevent cartilage degeneration in a murine OA model.

Objectives

We performed a systematic review of the literature to determine the safety and efficacy of bone morphogenetic protein (BMP) compared with bone graft when used specifically for revision spinal fusion surgery secondary to pseudarthrosis.

Thanks to recombinant DNA technique it is now possible to synthesise recombinant human osteogenic protein 1 (rh OP1), one of the best documented osteogenic proteins. This protein, linked to type 1 collagen as carrier, is the first drug with proven osteoinductive and osteoconductive properties approved for clinical use.

Osigraft (the commercial name of OP-1 and collagen) is also biocompatible, bioresorbable and lacks the risk of disease transmission. In the most challenging non-union, tibial non-union, the drug showed 80% efficacy as autograft with a better tolerability (i.e. lack of donor site complications) also in patients with previously failed autograft. Higher rates of success were also reported in recalcitrant long bone non-unions, i.e. a mean of 5 years of length of disease, incresaed number of previous surgeries.

Osigraft, also considering its physical characteristics, has to be implanted during the surgical procedure, with direct positioning at the non-union site; furthermore, its use is contraindicated in cases of infection of bone and infection/poor condition of surrounding soft tissues.

In our department we have treated up to now five patients with complex non-union of femoral neck and tibia; in three cases serious soft tissue conditions were present (crushing, infected necrosis) and one patient required plastic surgery. In all case we registered complete clinical and radiological healing after 3.5–7 months.

We also describe two cases of closed application of Osigraft, pushing the drug into the non-union site via a holed cannulated nail with the help of a probe under radioscopic control.

*Winner of ISFR Young Investigator Award

Purpose: The aim of this study was to compare the temporal expression pattern of factors related to cartilage and bone formation and endochondral ossification during standard and delayed bone healing for a more in-depth understanding of the molecular basis of disturbed bone healing and to elucidate suitable timing for substitution of factors to stimulate the healing process.

Methods: A tibial osteotomy was performed in two groups of sheep (n=30 each) and stabilized with either a rigid external fixator leading to standard healing or with a mechanically critical one leading to delayed healing. Hematoma/callus tissue was harvested 4, 7, 14, 21 and 42 days postop. qPCR was employed to determine the expression patterns of BMPs and other molecules.

Results: Gene expressions of BMP2, BMP4, BMP7, Noggin, MMP9 and MMP13 were distinctly lower in the delayed compared to the standard healing group at several time points from day 14, whilst no differential gene expression of Coll II and Coll X was found between both groups. Among the BMPs, BMP7 showed the most markedly differential expression. The first evident difference in BMP7 expression between both groups was found at day 14 suggesting that exogen substitution in the context of a therapeutic approach should be postponed. The differential expression pattern of both MMP9 and MMP13 suggests that there might be a failure or delay in endochondral ossification in delayed bone healing.

Conclusion: Downregulation in gene expression of osteogenic BMPs and cartilage matrix degrading MMPs may account for a considerable delay of bone healing.

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

Wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of RANL in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particletreated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis

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

Background. Bone Morphogenetic Protein (BMP) has been used in clinical practice to stimulate fracture healing and spinal arthrodesis. Difficulty in localising and maintaining BMP at the target site has resulted in the use of large doses of BMP, and has been associated with significant adverse effects. We have previously shown clay hydrogels can bind growth factors for localised efficacy. We hypothesised that localisation of BMP within clay gels would reduce the dose required to mediate bone formation. Methods. 2×10-4mg and 1×10-5 mg BMP were mixed in Laponite and applied to collagen sponge. 3 sponges containing high dose, and 3 containing low dose BMP were implanted subcutaneously in a mouse. This process was repeated in 8 mice, for controls, alginate hydrogel was used in a further 8 mice, and 1 mouse received 6 blank collagen scaffolds. Micro Computed Tomography was used to assess bone formation fortnightly; at 8 weeks the mice were culled and underwent histological analysis. Results. Mean Bone Volume formed within collagen per μg BMP was significantly greater with Laponite and low dose BMP compared to Alginate and Laponite with high dose BMP (p<0.0001). No bone formation was observed with Alginate and low dose BMP. Conclusions. We have demonstrated that Laponite is able to reduce, by several orders, the effective dose of BMP required to mediate ectopic bone formation compared to current gold standard methods of BMP delivery. Clinical translation of this finding offers, potentially, great significance to orthopaedic surgery. Level of Evidence. In vivo study. Approval. Our study received ethical approval complied with Home Office licensing. Acknowledgments. Funded by grants from EU(FP7) Biodesign, Rosetrees Trust, BBSRC and EPSRC

The purpose of this study was to quantify the effect of BMP 7 and recombinant Human (rh) BMP 2 at stimulating bone formation and bone union in trauma and elective orthopaedic surgery. Methods. We retrospectively reviewed the use of BMP 7 and rhBMP 2 at the Queen Alexandra Hospital between 2005 and 2012. The minimum follow up was three months. Inclusion criteria consisted of all patients who had failed to achieve previous surgical bone union and then received either BMP 7 or rhBMP 2 in an attempt to achieve bone union (as part of revision surgery). Patients who have not completed a minimum of three months follow up were excluded. Bone union was defined clinically and radiographically. Results. 17 patients were included (9 elective and 8 trauma patients). 9 patients received BMP2 (8 trauma and 1 elective) and 8 received rhBMP 2 (all elective). The average number of attempted fusions with autogenous bone graft prior to the use of BMP agent in the trauma and elective group was 2 and 3 respectively. The overall union rate following the use of BMP was 94.1%. 1 patient from the BMP 7 group (trauma patient) failed to unite. The union rate with BMP 7 and rhBMP 2 was 88.9% and 100% respectively. Following the use of BMP the average time to union was 117 days (BMP 7 124 days and rhBMP 2 112 days). Conclusion. Our results suggest both BMP 7 and rhBMP 2 to be an effective means of stimulating bone formation and bone union in patients with established non-union, in both trauma and elective orthopaedic surgery