The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.

Objectives

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis.

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.

Summary Statement. Treatment of non-union is a highly demanding field with respect to bone healing. BMP 7 is a useful, wide-ranged tool in treating non-union of the foot and benign bone tumors. It represents a low-risk procedure with a high level of reliability. Introduction. Treatment of non-union is a highly demanding field with respect to bone healing. Treatment of tibial fracture non-union with the bone morphogenetic protein 7 (BMP-7) has been successfully reported. BMP 7 is a recombinant human protein produced in ovary cells of the Chinese hamster. It is responsible for the differentiation of mesenchymal stem cells from the periost, muscle and sponious bone and stimulates bone formation. It is the aim of our study to investigate the use of BMP 7 for other locations than the tibia, such as the foot and benign bone tumors. We strive for union or revision in each medical case. Patients & Methods. At our clinic we applied BMP-7 to 13 patients (9 patients with non-union, 4 patients with benign bone cysts). 9 patients with non-union of the foot (4 forefoot, 1 midfoot, 3 hindfoot, 1 tibia) were surgically treated by resection, stabilisation, and application of BMP 7. The study included 5 men and 4 women at an average age of 58,4 years (range 33 – 80), 13 previous surgeries had been carried out. The period of follow up was on average 16.3 months (5 – 40 months). The indication for using BMP-7 instead of autologous bone graft was poor local blood supply, poor local soft tissue because of previous interventions and risk factors like smoking and diabetes. Following an indicated open biopsy, the 4 cases of benign bone tumors (1 juvenile bone cyst of the talus, 1 osteofibrose dysplasia of the proximal tibia and 2 juvenile bone cysts of the proximal humerus) were all treated with resection, followed by an application of BMP-7 and external or internal fixation. In addition two received bone grafting and two received cortisone. The average age of the tumor group was 16,75 years (11–24 years, 2 male, 2 female). Results. At follow-up all patients were satisfied with respect to pain and function, no operative complications had occurred and bone fusion had finished in 7 patients after 3 months. One ankle joint had a fibrous fusion but was free of pain. One arthrodesis of the first metatarsophalangeal joint was turned into a resection arthroplasty, today the patient is free of pain and uses a normal shoe. Both bone cysts have the radiological evidence of rehabilitation. At one humeruscyst we removed the TENS-nails without complications. We had no complications like heterotopic ossification, local erythema or pressure sensitivity. Discussion/Conclusion. These results show that BMP 7 is a useful, wide-ranged tool in treating non-union of the foot and benign bone tumors. It represents a low-risk procedure with a high level of reliability

This preliminary study evaluates a combination of bone morphogenetic protein (BMP)-7 and non-vascularised autologous fibular grafting (AFG) for the treatment of osteonecrosis of the femoral head.

BMP-7/AFG combination was applied in seven pre-collapse femoral heads (five Steinberg stage II, two stage III) in six patients. Pre- and post-operative evaluation included clinical (Harris hip score (HHS), visual analogue scale (VAS) for pain) and radiological assessment (radiographs, quantitative CT) at a mean follow-up of 4 years (2 to 5.5).

A marked improvement of function (mean HHS increase of 49.2) and decrease of pain level (mean VAS decrease of 5) as well as retention of the sphericity of the femoral head was noted in five hips at the latest follow-up, while signs of consolidation were apparent from the third post-operative month. One patient (two hips) required bilateral total hip replacement at one year post-operatively. In the series as a whole, quantitative-CT evaluation revealed similar densities between affected and normal bone. Heterotopic ossification was observed in four hips, without compromise of the clinical outcome.

In this limited series AFG/BMP-7 combination proved a safe and effective method for the treatment of femoral head osteonecrosis, leading to early consolidation of the AFG and preventing collapse in five of seven hips, while the operative time and post-operative rehabilitation period were much shorter compared with free vascularised fibular grafts.

Cite this article: Bone Joint J 2014;96-B:31–5.

Statement of purpose. To determine the outcome of the use of Bone Morphogenetic Protein 7 (BMP7) as a replacement for bone graft in a limb reconstruction unit. Methods. Retrospective case note and imaging review was performed on a cohort of 71 consecutive patients from October 2009 to October 2012 in whom BMP7 was used to achieve union. The patients were identified from a pharmacy database. Factors analysed included the perceived indication, location in the skeleton, age, comorbidities, type of procedure (non-union, fusion, docking site etc), complications and need for revision surgery. Results. BMP7 was used in 71 patients with a median age of 63 (mean 58, range 17–86). The majority of patients were in the seventh decade. 62 were non-unions, eight were for fusion of docking site and one for fusion of an osteotomy. Circular external fixation was used in 20, IM nails in four, and plate fixation in 47. Indications for using BMP7 as opposed to bone graft included advanced age, immunosuppressive comorbidities or medication and obesity. In 65 cases bony union was achieved after the index procedure and one patient needed further surgery. There were five failures: two patients died before union and three failed to heal with the index intervention, leading to 74 procedures in 71 patients. BMP7 was used in several anatomic sites: femur (n=29), humerus (n=15), tibia (n=18) and in fusion of a joint (n=9). Overall success rate for achieving union was 93%. Local inflammation lasting a mean of three weeks was seen in six cases, and five patients developed heterotopic ossification, of which one was symptomatic. Conclusion. BMP 7 can be used as a replacement for autologous bone graft with predictable success in situations where bone graft harvest may be deemed unsuitable either because of local or systemic factors

Purpose: Extracortical bone bridging and ingrowth have been shown to reduce stresses on the stem and cement mantle of tumor endoprostheses. The purpose of this study was to assess the effect of bone morphogenetic protein 7 (BMP-7) delivered by Peri-Apatiteâ (PA, Stryker Orthopaedics) hydroxyapatite coating on porous segmental replacement prostheses. Method: Eighteen mature mongrel canines were implanted with unilateral segmental replacement prostheses made of a cobalt-chromium (Co-Cr) alloy and coated with two layers of sintered Co-Cr alloy beads (diameter 600 to 800mm). The control group consisted of a plain porous coated segmental prosthesis without any PA coating. Group 2 consisted of a PA-coated segmental prosthesis coated with buffer solution. Group three consisted of a PA-coated segmental prosthesis loaded with rhBMP-7 (Stryker Biotech) in a buffer solution carrier. Group 1 had the implant only. Group 2 had the buffer solution evenly applied to the porous coat and group 3 had 2.9 mg of BMP-7 in liquid buffer solution evenly applied. The canines were allowed to fully bear weight without restrictions. The femurs were retrieved at twelve weeks for radiographic and histologic analysis. Results: Gross and radiographic data of the retrieved specimens showed that all six PA-coated implants augmented with BMP-7 had complete bone bridging; only one of the PA-coated implants and only two of the plain porous implants were completely bridged. There was a greater percentage of bone apposition for the BMP-7 augmented PA-coated group compared to both the plain (p=0.0026) and the PA-coated (p=0.0001). There was no difference in bone formation or bone apposition between the plain and PA-coated groups. Histology revealed greater depth of bone ingrowth in the BMP-7 augmented PA-coated group as compared to the plain (p< 0.0001) and the PA-coated (p< 0.0001) groups. There was also significantly greater bone apposition in the BMP-7 augmented PA coated groups as compared to the plain (p=0.0014) and PA-coated (p=0.0067) groups. There was no significant difference in depth of bone ingrowth or bone apposition between the plain and PA-coated groups. Conclusion: BMP-7 when used to augment PA-coated prostheses in a canine segmental defect model can significantly improve extracortical bone bridging and bone ingrowth. PA-coated implants may be considered to deliver the exogenous biological growth factors

To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approaches, controllable differentiation of mesenchymal stem cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. Although bilayered scaffolds have been investigated in vitro and in vivo, no culture system is available to test BMSCs differentiation into bone and cartilage simultaneously in bilayered scaffolds. This study investigated a defined culture media, which supported osteoblast and chondrocyte differentiation depending on growth factors implemented in biomaterials. In 2-dimensional culture, BMSCs differentiated to chondrocytes when transforming growth factor-beta 3 (TGF-β3) was added to the defined media, whereas osteogenic differentiation was induced by adding bone morphogenetic protein 7 (BMP-7). BMSC differentiation to osteogenic and chondrogenic lineages was further strengthen in 3-dimensional culture. Proteoglycan formation, type II collagen, and aggrecan were upregulated in the defined media when BMSCs were mixed with fibrin gel impregnated with TGF-β3. Mineralization and the expression of osteogenic markers such as alkaline phosphatase, osteopontin, and osteoclacin were noticeable when BMSCs cultured in hydroxyapatite-tricalcium phosphate (HA/TCP) scaffolds coated with BMP-7. This study generated and tested a growth media, which could induce osteogenic and chondrogenic differentiation of BMSCs in one culture system. These results will help the development of tissue substitutes for multi-complexed tissues such as subchondral replacement

Heterotopic ossification occurring after the use of commercially available bone morphogenetic proteins has not been widely reported. We describe four cases of heterotopic ossification in patients treated with either recombinant bone morphogenetic protein 2 or recombinant bone morphogenetic protein 7. We found that while some patients were asymptomatic, heterotopic ossification which had occurred around a joint often required operative excision with good results

Recombinant Bone Morphogenetic Protein 7 (OP-1) has been available in the UK since 2001, but there has been little published data on its use in the upper limb. In our institution OP-1 has been used in the management of 23 upper limb patients between 2001–2005, including 10 humeral non-unions. We believe this represents one of the largest single-unit cohorts of humeral fractures treated with OP-1. We reviewed the 10 humeral patients, who were all tertiary referrals with established non-unions. Two had been treated non-operatively before referral. The remaining eight had undergone a mean of 2.1 operations before OP-1 was used, with autologous bone grafting used in the majority of cases. Surgery occurred at a mean of 70.5 months following initial fracture. Seven patients underwent revision of the fracture fixation, and autologous bone graft was used with the OP-1 in 8 cases. Clinical union was established in 8 patients (80%) within a mean of 7.4 months. Radiological union was achieved in 8 patients (80%) within a mean of 9.1 months. No complications or adverse effects from the use of OP-1 were encountered. Both cases which failed to unite had a definite history of deep infection prior to index surgery, despite initial treatment with a staged revision procedure before OP-1 use. This study shows that OP-1 can be used successfully in the treatment of recalcitrant non-unions of the humerus following failure of traditional fracture management methods

Bone Morphogenetic Protein 7 (BMP7) is a powerful osteoinductive substance that could stimulate bone formation in difficult conditions including distraction osteogenesis. However, to be effective, large unphysiological doses are required. Blocking the expression of BMP antagonists could amplify the effects of BMP7, allowing smaller doses of BMP7 to be used without altering its osteogenic potential. In this study, BMP7 antagonist Noggin was shown to be upregulated following BMP7 injection in a rabbit distraction osteogenesis model suggesting a role for Noggin in controlling BMP7 activity. Blocking Noggin expression may thus permit smaller doses of BMP7 to be used effectively. Distraction osteogenesis (DO) is an excellent method to form new bone. However, the long duration the external fixator has to be kept on until the new bone consolidates, could lead to numerous problems. BMP7 may accelerate bone formation in DO. However, large doses of BMP7 may be necessary. In this study, we investigated the expression of BMP7 antagonist Noggin in DO. Noggin may control BMP7 activity through a negative feedback mechanism. Blocking Noggin may amplify the effects of BMP7, thus permitting the use of smaller doses of BMP7 effectively in DO. Using smaller doses of BMP7 – while maintaining its powerful effects – may decrease side effects and render this drug more affordable economically. Noggin is normally expressed in DO. Its expression is upregulated by local application of BMP7. Its expression is co-localized to the same cells that express BMP7 and its receptors. The right tibia of sixteen rabbits was lengthened using a uniplanar fixator. The rabbits were divided into two groups: one received seventy-five micrograms recombinant BMP7 and the other placebo. All injections were performed one week after start of distraction. Rabbits were sacrificed ten minutes, one day, two days and two weeks following the injections. The expression of Noggin was studied in the distracted tissue by immunohistochemistry. Noggin may play a role in DO. Blocking its action may have huge clinical implications, by permitting the use of smaller – but equally effective – amounts of BMP7. Funding: CIHR, FRSQ and Shriners of North America

The different pathways by which bone morphogenetic protein 7 (BMP-7) could exert its osteogenic function in distraction osteogenesis (DO) were investigated. Using immunohistochemistry, the temporal and spatial expression of markers for angiogenesis, cell proliferation, Indian hedgehog pathway, osteogenic growth factors and their receptors were investigated in a rabbit model of DO. Our results showed that local injection of BMP-7 at the lengthened site caused up-regulation of expression of growth factors and their receptors, cell proliferation and vascular markers and Indian hedgehog gene in a temporal fashion. By knowing these pathways, manipulation of DO by pharmaceutical agents may be possible. Based on preliminary data, BMP-7 can accelerate the consolidation of newly formed bone if locally injected early in the distraction phase; however, the exact mechanism remains unknown. The purpose of this study was to investigate the different pathways through which BMP-7 exerts its effects in DO. The right tibia of twenty-four rabbits was lengthened 2.0 cms. The rabbits were divided into three groups : control, placebo and treated groups. The rabbits received no injection (control), buffer (placebo) and 75 micro grams BMP7 (treated) in the distracted zone one week after the start of distraction. The rabbits were sacrificed ten minutes, one day, two days and two weeks following the injections. Using immunohistochemistry, the different pathways of bone formation were assessed by analysing the expression of markers for angiogenesis (VGEF, Vascular Endothelial Growth Factor and PECAM , platelet endothelial cell adhesion molecule) , cell proliferation markers (PCNA, proliferation cell nuclear antigen), osteogenic growth factors (TGFβ, IGF, FGF and their receptors) and Indian hedgehog gene as part of the parathyroid hormone related peptide pathway. BMP-7 may stimulate bone formation through several pathways in a temporal fashion early after local injection, by up-regulating the expression of numerous osteogenic growth factors and their receptors and Indian hedgehog, and late two weeks after the injection, by up-regulating cell proliferation and vascular markers. Our results showed the possible mechanisms of action of BMP-7 in DO and more importantly the various pathways through which pharmacological agents could be used in the manipulation of DO

We describe a 13-year-old boy with atrophic tibial pseudarthrosis associated with neurofibromatosis who had undergone nine unsuccessful operations. Eventually, union was obtained by the use of bone morphogenetic protein 7 in conjunction with intramedullary stabilisation and autologous bone graft

There can be no doubt that bone morphogenetic proteins play a hierarchic role in the osteogenetic cascade. Pre-clinical and clinical trials have confirmed their decisive role in achieving anterior lumbar fusion, as they direct mesenchymal stem cells toward osteoblastic lineages.The present study is concerned with initial experience in the application of autologous mesen-chymal stem cells and various growth factors (BMP-7,VEGF,TGFbeta) in the treatment of paediatric spinal pathologies. Eleven patients affected by serious forms of congenital infantile scoliosis, idiopathic scoliosis and grade I spondylolisthesis received surgical treatment. In three patients with congenital infantile scoliosis, ages ranging from 3 to 12 years, the surgical procedure was an anterior and posterior fusion at the level of the hemiver-tebra, extending it to a level above and below it by means of in situ decortication of the vertebral plates and laminae on the convex side and delivery of stem cells taken from the iliac bone and applied in situ by means of bovine collagen sponge (Healos system). No fixation device was added. Plaster and brace were used during the postoperative course for 9 months. In two cases of intertransverse in situ fusion for grade I spondylolisthesis the age of the patients was 13 and 16 years, respectively, and the surgical procedure consisted in the standard technique to which was added delivery of a mixture composed of small bone chips obtained from decortication, 3.5 mg of eptotermin alpha (Op-1, BMP7), and autologous stem cells taken from iliac bone. A special form of informed consent was obtained for these two patients because of their incomplete bone maturity. TLSO was used in the postoperative course for 2 months. In the six patients with idiopathic scoliosis, ages ranged from 13 to 15 years and the treatment consisted in posterior instrumentation and fusion by means of rods, transpedicular screws and hooks; standard fusion techniques were supported by local bone chips obtained from decortication, placed on collagen sponges and combined with autologous stem cells taken from iliac bone with the addition of platelet gel derived from the autologous preoperative blood collection. No bone chips were taken from iliac wing. The results were evaluated by X-rays and CT at intervals of 1, 2, 4, 6 and 12 months. In the cases of congenital scoliosis a solid fusion area was obtained only for posterior hemiephysiodesis, without a parallel bone signal of fusion at the anterior level. In the cases of intertransverse in situ fusions for spondy-lolisthesis there was a beginning fusion already visible on the first X-ray control 1 month postopoeratively, confirmed at successive check-ups and maintained in the follow-up. The cases of idiopathic scoliosis showed an initial ossification of the grafts and signals of fusion at the 6-month check-up. The isolated use of stem cells, although promising from a theoretical point of view, did not prove encouraging in the cases of anterior fusion. It is most probable that the absence of instrumentation induced a defect in the stable fixation of the fused segments, the latter being a crucial factor.The cases of in situ fusion for spondylolisthesis confirm the hierarchic role of the bone morphogenetic protein 7 in inducing the mesenchymal stem cells, released in situ from decortication, toward osteoblastic lineage. To our knowledge these represent the first two cases of use of OP-1 in patients younger than 18 years. In the fusion areas which are more extensive in length (idiopathic scoliosis) the added use of autologous stem cells mixed with platelet gel seems to improve the physiological processes of fusion. It will be necessary to monitor the long-term results of these procedures with special regard to loss of correction and weakness of the fusion area causing torsional or flexion-extension stress. These possible costs have to be compared with the verified benefits of a better use of blood in its capacity to save on corresponding haemotransfusion, combined with the absence of comorbidity related to the donor site of iliac bone chips

Aim: Mesenchymal stem cells (MSCñs) attach to hydroxyapaptite surfaces (HA) surfaces and given appropriate stimuli from human Bone Morphogenetic Protein 7 (OP -1), will differentiate into osteogenic cells. Our hypothesis is that combining HA/MSC/BMP-7 will provide a superior osseoinductive property compared to HA alone. Methods: Porous hydroxyapatite (74.6% porosity, and 0.65% closed porosity) loaded with MSCñs (2 x 105) were compared to samples loaded with rhBMP-7 (400 ng/0.1g HA,) of the same MSC concentration over a fourteen day period. Quantitative analysis (Cell proliferation, measured by total DNA and the Alamar blue assay and Cell differentiation- alkaline phosphatase activity) and qualitative (Light and Scanning Electron Microscopy) were performed. The Students T-test was performed. Ethical approval for the use of human tissue was obtained prior to experimentation Results: Cell proliferation as indicated by total DNA, and Alamar blue was signiþcantly enhanced (P< 0.05) in the BMP-7 loaded composite at all time points. ALP production and release was enhanced in loaded samples. ALP production per unit DNA was also enhanced in the loaded samples and was signiþ-cant at day fourteen. Conclusion: Results indicate that the loaded composites showed enhanced cell proliferation, and ALP production and release. SEM analysis also demonstrated enhanced cell attachment and an increase number of proliferative cells. Thus the HA/MSC/BMP-7 composite displayed superior osseo-inductive properties in comparison to the HA/MSC composite