Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model. A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site. In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity. This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem

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

Objectives. The injured anterior cruciate ligament (ACL) is thought to exhibit an impaired healing response, and attempts at surgical repair have not been successful. Connective tissue growth factor (CTGF) is reported to be associated with wound healing, probably through transforming growth factor beta 1 (TGF-β1). Methods. A rabbit ACL injury model was used to study the effect of CTGF on ligament recovery. Quantitative real-time PCR (qRT-PCR) was performed for detection of changes in RNA levels of TGF-β1, type 1 collagen (COL1), type 2 collagen (COL2), SRY-related high mobility group-box gene9 (SOX9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase 13 (MMP-13). Expression of related proteins was detected by Western blotting. Results. The current study showed that CTGF could promote the recovery of an injured anterior cruciate ligament. It can upregulate mRNA and expression of TGF-β1, COL1, COL2, SOX9, and tissue inhibitor of TIMP-1, and downregulate mRNA and expression of MMP-13, suggesting that the curative effect of CTGF on injured rabbit ligaments is through regulation of these cellular factors. Conclusions. This finding revealed the healing role of CTGF in injured tissues and provides new possibilities of treating injured tissues and wound healing by using CTGF. Cite this article: X. Sun, W. Liu, G. Cheng, X. Qu, H. Bi, Z. Cao, Q. Yu. The influence of connective tissue growth factor on rabbit ligament injury repair. Bone Joint Res 2017;6:399–404. DOI: 10.1302/2046-3758.67.BJR.2016-0255.R1

Purpose. The aim of this study was to investigate whether growth factors essential for fracture healing are released in the immediate aftermath following fracture and whether reaming of IM cavity causes increased liberation of these autocoids. Methods. Consecutive adult patients with femoral shaft fractures forming two groups (a group who received unreamed nail (n=10) and a second group who received reamed nail (n=10) were recruited for this study. Peripheral blood samples and samples from the femoral canal before and after reaming and before and after the solid nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor I (IGF-I) Transforming Growth Factor beta 1 (TGF-. 2. 1) and BMP-2 levels was measured. Results. In total 20 patients were studied. The mean age was 38 years (range 20-63). Reaming substantially increased all studied growth factors locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-1 was increased by 31.5% and TGF-b1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF and TGF-. 2. 1 were not changed while the levels of IGF-I were decreased by 10%. The levels of these factors in peripheral circulation were not altered despite the technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. Conclusion and significance. This study indicates that reaming of IM Canal is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules

Purpose: The aim of this study was to investigate whether growth factors essential for fracture healing are released in the immediate aftermath following fracture and whether reaming of IM cavity causes increased liberation of these autocoids. Methods: Consecutive adult patients with femoral shaft fractures forming two groups (a group who received unreamed nail (n=10) and a second group who received reamed nail (n=10) were recruited for this study. Peripheral blood samples and samples from the femoral canal before and after reaming and before and after the solid nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor I (IGF-I) Transforming Growth Factor beta 1 (TGF-. 2. 1) and BMP-2 levels was measured. Results: In total 20 patients were studied. The mean age was 38 years (range 20–63). Reaming substantially increased all studied growth factors locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-1 was increased by 31.5% and TGF-b1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF and TGF-. 2. 1 were not changed while the levels of IGF-I were decreased by 10%. The levels of these factors in peripheral circulation were not altered despite the technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. Conclusion and Significance: This study indicates that reaming of IM Canal is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules

Summary. We found an increased natural expression of the growth factors bFGF, BMP-12, VEGF, and TGF-b1 during tendon healing of rat Achilles tendons. External application of these growth factors improved the tendons failure load in the early healing phase. Introduction. Tendon ruptures recover slowly and the healing of injuries can be devastating. Growth factors are known to influence tendon healing. However, only little is understood about growth factors in a healing tendon. Aim of this study was to investigate the influence of growth factors on tendon healing of rats following their natural expression. Methods. The Achilles tendon of rats were transected and resutured. First the expression of bFGF, BMP-12, VEGF and TGF-b1 was assessed by immunohistochemical analysis 1 to 8 weeks after surgery. Second the maximal failure load of healed Achilles tendons was measured dependent on the external application of bFGF, TGF-b1 and BMP-12. Results. The natural expression of bFGF (p<0.05), BMP-12 (p<0.05), TGF-b1, and VEGF was highest 1 week after transection. VEGF expression persisted during the remaining period whereas bFGF and BMP-12 declined. TGF-b1 expression peaked again after 8 weeks. A combined application of bFGF, TGF-b1 and BMP-12 resulted in a 4fold greater load to failure after 1 week (p<0.05), whereas a sequential treatment of these growth factors increased the load to failure even 5.5fold (p<0.05). Conclusion. During natural tendon healing the growth factors bFGF, BMP-12, VEGF, and TGF-b1 are differentially expressed. Additional administration of the mentioned growth factors can improve the load to failure in the early healing phase of rat Achilles tendons

Injury to muscles is very common. We have previously observed that basic fibroblast growth factor (b-FGF), insulin growth factor type 1 (IGF-1) and nerve growth factor (NGF) are potent stimulators of the proliferation and fusion of myoblasts in vitro. We therefore injected these growth factors into mice with lacerations of the gastrocnemius muscle. The muscle regeneration was evaluated at one week by histological staining and quantitative histology. Muscle healing was assessed histologically and the contractile properties were measured one month after injury. Our findings showed that b-FGF, IGF and to a less extent NGF enhanced muscle regeneration in vivo compared with control muscle. At one month, muscles treated with IGF-1 and b-FGF showed improved healing and significantly increased fast-twitch and tetanus strengths. Our results suggest that b-FGF and IGF-1 stimulated muscle healing and may have a considerable effect on the treatment of muscle injuries

INTRODUCTION: Tissue growth factors have been extensively investigated as agents for acceleration of wound repair. Individual recombinant molecules have shown promise in animal models, but in humans both safety and efficacy remain questionable. 1,. 2. and costs are substantial. Recently introduced technologies allow intra-operative collection of the full naturally occurring array of tissue growth factors contained in platelets and white cells. 3. These preparations rely on the normal healing cascade for their performance, and their activity is limited solely to the wound site without undesirable proliferative or inappropriate tissue formation and no systemic effects. Numerous methodologies have been proposed for intra-operative preparation of autologous tissue growth factors for topical application, and an ever widening variety of approaches and formulations are available to the practitioner. 3. Physicians trying to decide which technique to adopt can easily find themselves bewildered while attempting to sift through myriad proponent’s claims. 4. PURPOSE: This presentation will review the state of the art, including: a summary of the role of autologous growth factors in bone fusion; a discussion of the importance of dosage and carrier matrix effects; an outline of the mechanics of intraoperative preparation; a survey of the capabilities of various techniques, and; an overview of in vitro, experimental and clinical studies. Audience members will receive a detailed understanding of the physiology, mechanics and clinical range of applicability for this newly emerging technology. This information will aid clinicians in choosing the most appropriate methodology for their practice

INTRODUCTION: Tissue growth factors have been extensively investigated as agents for acceleration of wound repair. Individual recombinant molecules have shown promise in animal models, but in humans both safety and efficacy remain questionable. 1,. 2. and costs are substantial. Recently introduced technologies allow intra-operative collection of the full naturally occurring array of tissue growth factors contained in platelets and white cells. 3. These preparations rely on the normal healing cascade for their performance, and their activity is limited solely to the wound site without undesirable proliferative or inappropriate tissue formation and no systemic effects. Numerous methodologies have been proposed for intra-operative preparation of autologous tissue growth factors for topical application, and an ever widening variety of approaches and formulations are available to the practitioner.3 Physicians trying to decide which technique to adopt can easily find themselves bewildered while attempting to sift through myriad proponent’s claims. 4. PURPOSE: This presentation will review the state of the art, including: a summary of the role of autologous growth factors in bone fusion; a discussion of the importance of dosage and carrier matrix effects; an outline of the mechanics of intraoperative preparation; a survey of the capabilities of various techniques, and; an overview of in vitro, experimental and clinical studies. Audience members will receive a detailed understanding of the physiology, mechanics and clinical range of applicability for this newly emerging technology. This information will aid clinicians in choosing the most appropriate methodology for their practice

Frozen shoulder is a chronic fibrosing condition of the capsule of the joint. The predominant cells involved are fibroblasts and myofibroblasts which lay down a dense matrix of type-I and type-III collagen within the capsule. This subsequently contracts leading to the typical features of pain and stiffness. Cytokines and growth factors regulate the growth and function of the fibroblasts of connective tissue and remodelling of the matrix is controlled by the matrix metalloproteinases (MMPs) and their inhibitors. Our aim was to determine whether there was an abnormal expression or secretion of cytokines, growth factors and MMPs in tissue samples from 14 patients with frozen shoulder using the reverse transcription/polymerase chain reaction (RT/PCR) technique and to compare the findings with those in tissue from four normal control shoulders and from five patients with Dupuytren’s contracture. Tissue from frozen shoulders demonstrated the presence of mRNA for a large number of cytokines and growth factors although the frequency was only slightly higher than in the control tissue. The frequency for a positive signal for the proinflammatory cytokines Il-1β and TNF-α and TNF-β, was not as great as in the Dupuytren’s tissue. The presence of mRNA for fibrogenic growth factors was, however, more similar to that obtained in the control and Dupuytren’s tissue. This correlated with the histological findings which in most specimens showed a dense fibrous tissue response with few cells other than mature fibroblasts and with very little evidence of any active inflammatory cell process. Positive expressions of the mRNA for the MMPs were also increased, together with their natural inhibitor TIMP. The notable exception compared with control and Dupuytren’s tissue was the absence of MMP-14, which is known to be a membrane-type MMP required for the activation of MMP-2 (gelatinase A). Understanding the control mechanisms which play a part in the pathogenesis of frozen shoulder may lead to the development of new regimes of treatment for this common, protracted and painful chronic fibrosing condition

Objectives. Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought. Methods. Primary human osteoblasts in in vitro culture were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1) or 48 hours (BMP2 and FGF2). Experiments were performed during the exponential growth phase with approximately 1e7 cells per 75 cm. 2. flask. mRNA was reverse transcribed directly and analysed using RT-PCR Taqman assays. Expression levels of key genes involved in cell growth and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A, LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific Taqman probes. . Results. Autocrine expression of BMP2 is stimulated by FGF2 and BMP2 itself. BMP2 and FGF2 act as proliferative factors as indicated by reduced expression of ALP and POSTN, whereas IGF1 exhibits a more subtle picture: the Wingless und Int-1 (Wnt) signalling pathway and the Smad pathway, but not p38 mitogen-activated protein (MAP) kinase signalling, were shown to be activated by IGF1, leading to proliferation and differentiation of the cells. . Conclusions. For future use of autologous bone cells in the management of bony defects, new treatment options take advantage of growth factors and differentiation factors. Thus, our results might help to guide the timely application of these factors for the expansion and subsequent differentiation of osteoblastic cells in culture. Cite this article: Bone Joint Res 2014;3:236–40

Recent clinical studies on targeting nerve growth factor (NGF) in chronic low back pain and knee osteoarthritis have demonstrated efficient pain reduction in a short-term treatment regimen. However, the increased risk for the development of rapid progressive osteoarthritis at the required high drug dose remains a serious concern and prompts thorough analysis of the tissue distribution and role of NGF in degenerative musculoskeletal disorders. Here, we sought to investigate tissue distribution of NGF, its high affinity receptor TrkA and CD68-positive macrophages in human facet joint osteoarthritis of the lumbar spine. Facet joint specimens (n=10) were harvested by facetectomy from patients undergoing elective lumbar intervertebral spine fusion. Facet joint osteoarthritis and presence of synovitis was graded using preoperative magnetic resonance imaging. Tissue distribution of NGF, TrkA and CD68 was determined using immunohistochemistry. Tissue degradation was graded on safranin-O-stained tissue sections. Association between imaging parameters and tissue distribution was determined using Pearson correlation analysis. Synovitis was present in 6 cases and facet joints displayed moderate to severe radiological osteoarthritis (median Weishaupt grade; 2 [1.5–3]). NGF was expressed in 8 of 10 specimens. NGF was expressed in connective tissue, articular and fibrocartilage, but not bone tissue. Cartilaginous NGF expression was predominantly found in the extracellular matrix of superficial cartilage tissue with complete loss of proteoglycans, chondrocyte death and structural damage (fissures). Loss of cartilage proteoglycan staining alone did not display NGF immunoreactivitiy. NGF expression was not correlated with radiological osteoarthritis severity or presence of synovitis. NGF high affinity receptor TrkA was exclusively expressed in bone marrow tissues. Differential grades of bone marrow infiltration by CD68-positive macrophages were observed, yet these were not associated with NGF expression. Targeting NGF in chronic low back pain and/or facet joint osteoarthritis might affect pathomechanisms in cartilaginous tissues and NGF signalling in the bone marrow compartment

Objectives. The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known. Methods. In this study, TGF-β1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-β1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours. Results. A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-β1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats. Conclusion. Mechanical stress-induced overexpression of TGF-β1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-β1 inhibitor can inhibit articular cartilage degradation. Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1). Bone Joint Res 2018;7:587–594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels. 2. to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis. Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies. Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.)

Aims: The intervertebral disc (IVD) consists of three structurally distinct areas; a nucleus pulposus (NP), annulus fibrosus (AF) and two cartilage endplates that together form a functional unit that allow flexibility of the spinal column and load transfer from adjoining vertebrae. The NP and AF contain cells that are phenotypically similar to chondrocytes found in articular cartilage. They also produce the 2 major matrix components aggrecan and collagen-type I and II. One feature of IVD degeneration is breakdown of the cartilage matrix. Using soluble growth factors could stimulate new matrix formation and help regenerate degraded discal cartilage. The aim of this study was to demonstrate the presence of four growth factor receptors within the IVD. Methods and Results: Using immunohistochemsitry, we targeted expression of four growth factor receptors, (BMPRII, FGFR3, IGFR-1 and TGFβII), in biopsies taken from normal and degenerate IVD. Receptor expression was scored across regions of the disc using a peer-reviewed system that assessed the proportion of cells expressing a particular antigen and the average level of expression for those cells. For FGFR3, IGFR-1 and TGFβII, cells of the outer part of NP and inner AF expressed significantly higher receptor levels. The expression BMPRII deviated from that pattern and was present at higher levels in the inner and outer NP than in the AF. Although there were significant differences between FGFR3 expression in normal and degenerate biopsies, that was not the case for the other receptors. Growth factor receptor expression was also detectable on the ingrowing neurons and blood vessels that characterise part of the disease aetiology. Conclusion: In conclusion, all of the receptors were found in the IVD, predominantly within the NP, suggesting that, addition of the ligands for these receptors may elicit a physiological response from disc chondrocytes

Aims: In recent years more and more studies tried to evaluate possible inßuences of different growth factors on hyaline cartilage regeneration. In a rabbit model, HGF (hepatocyte growth factor) was proven to increase the amount of hyaline-like chondrocytes in a mixed þbrocartilaginous regenerate of small defects. The present study was undertaken to evaluate, whether intraarticular administration of hepatocyte growth factor inßuences the ingrowth of osteochondral grafts in a sheep model. Methods: Both knee joints of a sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral compartment. The sheeps were divided into two groups. In one group hepatocyte growth factor was administered by intraarticular injections given three times a week for four weeks. The control group received isotonic sodium chloride injections. The animals were sacriþced after three months and the received knee joints were evaluated histologically. Results: Histological evaluation showed that the autologous osteochondral grafts were healed in at the level of the subchondral bone. A healing or ingrowth at the level of the cartilage could not be observed. Anyway, histological evaluation of the transplanted grafts according to Mankin showed, that the cartilage of the HGF group showed less signs of degeneration than the control group. In the HGF group less cloning of chondrocytes and less irregularities of the articular surface were observed. Conclusion: In conclusion, HGF positively inßuenced the structure of the transplanted osteochondral graft, but could not diminish the þssures in the marginal zone of the grafts

Due to well-known disadvantages of the autologous bone graft, many alternatives have been studied for a reliable spinal fusion. Herein, we aimed to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. 36 male SD rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: Sham control group, Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) group and HA/β-TCP + EGF group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p=0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p=0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42– 0.59), 0.73 Nmm (0.49– 0.88) and 0.91 Nmm (0.66– 1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p=0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p=0.087). This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates

Stimulation of bone healing and bone formation through local application of growth factors may improve the clinical outcome in high tibial osteotomy in varus knee. The goal of the present study is to evaluate the effectiveness of autologous growth factors asscociated with or without granular coralline hydroxyapatite used to fill open wedge defects after tibial osteotomy for valgisation in 13 patients with medial femorotibial degeneration. The integration of the coralline hydroxyapatite was complete in every case; we did not encounter any general or local problems, nor were there any intra-operative, peri-operative or post-operative complications. We conclude that local application of autologous growth factors associated with coralline hydroxyapatite is a good solution to stimulate callus formation and ossification in the early phase of bone healing and can substitute bone graft to fill bone defects

We studied the presence of anabolic growth factors in human herniated intervertebral discs (IVD) using a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Messenger RNA (mRNA) was isolated from the nucleus pulposus using oligo (dT). 25. superparamagnetic beads and probing with gene-specific primers in RT-PCR. mRNA coding for TGF-α (3/10), EGF (0/10), TGF-β1 (0/10) and TGF-β3 (2/10) or the EGF receptor (EGF-R; 0/10) and TGF-β type-II receptor (0/10) was found only occasionally. Beta-actin was always present and positive sample controls confirmed the validity of the RT-PCR assay. These RT-PCR findings were confirmed using immunohistochemical staining of EGF and TFG-β, whereas TGF-α protein was always found associated with discocytes. We conclude that the nucleus pulposus of the herniated IVD is vulnerable to proteolytic degradation and depletion of proteoglycans due to the lack and/or low production of anabolic growth factors/receptors which could increase the local synthesis of the extracellular matrix

Glenoid replacement is technically challenging. Removal of a cemented glenoid component often results in a large osseous defect which makes the immediate introduction of a revision prosthesis almost impossible. We describe a two-stage revision procedure using a reversed shoulder prosthesis. Freeze-dried allograft with platelet-derived growth factor was used to fill the glenoid defect. Radiological incorporation of the allograft was seen and its consistency allowed the placement of a screwed glenoid component. There were no signs of new mature bone formation on histological examination. The addition of platelet-derived growth factor to the allograft seems to contribute to an increase in incorporation and hardness, but does not promote the growth of new bone

The reconstruction of bone defects with biomaterials represents a potential alternative to the transplantation of autologous and allogenic bone. Ceramic materials can be combined with growth factors (i.e. BMPs) to render them osteoinductive. Coating of biomaterials with growth factors has mostly been attempted by adsorption onto the material’s surface. The superficial deposition usually results in an immediate passive release of the proteins, thus restricting their temporal availability during bone healing. It was hypothesized that a co-precipitation of proteins onto calcium phosphate ceramics may provide the possibility to achieve a prolonged release of proteins from the material without impairing the biologic activity of growth factors. Tritium labelled bovine serum albumin ([3H]BSA) and recombinant human BMP2 (rhBMP2) were coated onto biphasic calcium phosphate (BCP) ceramics using a coprecipitation technique of proteins together with calcium phosphate (Liu Y et al. 2001). The co-precipitation was compared to conventional adsorption of proteins to ceramic materials. The passive and cell-mediated release of [3H]BSA was investigated during 19 days. To analyze the cell-mediated protein release, murine bone marrow cells were seeded onto ceramics and differentiated to osteoclasts or to monocytes/macrophages. To assess whether rhBMP2 co-precipitated to BCP ceramics retained its biologic activity the growth factor’s ability to induce the differentiation of primary murine osteoblasts was studied. After 19 days 71.7±5.3% of the adsorbed [3H]BSA was passively released (63.0±6.0% within 4 days). The passive liberation of [3H]BSA was effectively reduced using the coprecipitation technique (12.5±2.0% within 19 days, 10.1±2.3% within 4 days, p< 0.001). Further analysis demonstrated a sustained, osteoclast-mediated release of coprecipitated [3H]BSA from calcium phosphate ceramics which was blocked by the addition of calcitonin. Passive release of adsorbed and co-precipitated BMP2 led to a temporally restricted stimulation of murine osteoblasts. Cell-mediated liberation of co-precipitated BMP2 induced a sustained stimulation of the differentiation of osteoblasts. The successful application of exogenously added growth factors depends critically on the mode of delivery. It has been shown that a sustained availability of BMP2 is beneficial for bone healing. Application of the co-precipitation technique resulted in a long-term release of proteins from BCP ceramics mediated by active resorbing osteoclasts without impairing the biologic activity of rhBMP2. Co-precipitating growth factors onto BCP ceramics provides a potential to shift the initial extensive liberation to a sustained release of bioactive proteins. This method of protein delivery may represent a possibility to achieve a more physiological availability of growth factors during bone regeneration

Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd

Nerve growth factor (NGF) is involved in several joint diseases. It participates in pain initiation, inadequate nociception and neurogenic inflammation; its concentrations are increased in synovial fluid and tissue from human and experimental arthritis. However, data about its role in normal and pathological articular cartilage are scant and conflicting. This study assesses the effects of different. NGF concentrations on cultured healthy human chondrocytes by evaluating cell proliferation, cell phenotype, and gene expression. The 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl-2H-tetrazolium bromide (MTT) test excluded an influence on cell viability; alcian blue and S100 staining demonstrated that NGF induced de-differentiation of the chondrocyte phenotype; real-time PCR disclosed that it reduced the expression of collagen type II (COL2A1) and transforming growth factor-β (TGF-β), key factors involved in articular cartilage integrity, and stimulated upregulation of metalloproteinase (MMP)-3 and MMP-13. These findings suggest that NGF may adversely affect differentiated chondrocytes from articular cartilage by inhibiting the expression of the collagens found in normal articular cartilage (COL2A1), while exerting a degradative effect though TGF-β downregulation and MMP-13 and MMP-3 upregulation. Further investigation is required to determine whether the gene expression pattern found in our study is associated with changes in protein expression

Introduction and purpose: We have studied the in-vitro response of older and osteoarthritic chondrocytes when confronted with various factors in order to analyze the possible reversion of their phenotype to that of healthy chondrocytes. Materials and methods: The study used cartilage from young (3 months’ old) and old (7 years old) lambs with an osteoarthritic pathology. The latter group was obtained by means of a meniscectomy after a two-month evolution. Cells coming from the femur cartilage were isolated by means of collagenase digestion and cultured in a single layer using a DMEM culture medium supplemented with 10% fetal serum, penicilin and streptomycin, hepes and L-cystein (Gibco-BRL®). BrdU incorporation assays were performed by means of an ELISA protocol in order to analyze the proliferation rate. Later, a gene expression analysis was conducted using RT-PCR. The treatment was carried out at a concentration of 50 ng/mL using FGFa, IGF-a, TGF-b (Peprotech Inc) and OP-1 (Stryker) growth factors, and later growth factor combinations FGFa/IGF-1, FGFa/OP-1, FGFa/TGF-b and TGF-b/OP-1. Results and conclusions: Comparison between the three groups showed that the proliferation rate was lower in older and osteoarthritic cells. These last two groups, however, did not have the same expression pattern as the genes studied. The analysis of the response to growth factors showed that FGFa and IGF-1 were the most efficient ones, and their combination proved to be the most powerful of all

Aims. It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway. Materials and Methods. An implant was inserted into the right tibia of 16-week-old female C57BL/6 mice (n = 38). Mice received anti-VEGF receptor-1 (VEGFR-1) antibody (25 mg/kg) and VEGF receptor-2 (VEGFR-2) antibody (25 mg/kg; n = 19) or an isotype control antibody (n = 19). Flow cytometric (n = 4/group) and immunofluorescent (n = 3/group) analyses were performed at two weeks post-implantation to detect the distribution and density of CD31. hi. EMCN. hi. endothelium. RNA sequencing analysis was performed using sorted CD31. hi. EMCN. hi. endothelial cells (n = 2/group). Osteoblast lineage cells expressing osterix (OSX) and osteopontin (OPN) were also detected with immunofluorescence. Mechanical pull-out testing (n = 12/group) was used at four weeks post-implantation to determine the strength of the bone-implant interface. After pull-out testing, the tissue attached to the implant surface was harvested. Whole mount immunofluorescent staining of OSX and OPN was performed to determine the amount of osteoblast lineage cells. Results. Flow cytometry revealed that anti-VEGFR treatment decreased CD31. hi. EMCN. hi. vascular endothelium in the peri-implant bone versus controls at two weeks post-implantation. This was confirmed by the decrease of CD31 and endomucin (EMCN) double-positive cells detected with immunofluorescence. In addition, treated mice had more OPN-positive cells in both peri-implant bone and tissue on the implant surface at two weeks and four weeks, respectively. More OSX-positive cells were present in peri-implant bone at two weeks. More importantly, anti-VEGFR treatment decreased the maximum load of pull-out testing compared with the control. Conclusion. VEGF pathway controls the coupling of angiogenesis and osteogenesis in orthopaedic implant osseointegration by affecting the formation of CD31. hi. EMCN. hi. endothelium. Cite this article: Bone Joint J 2019;101-B(7 Supple C):108–114

We undertook a prospective study to evaluate the prognostic significance of the serum levels of vascular endothelial growth factor (VEGF) in predicting the survival of patients with osteosarcoma. The levels were measured by an enzyme-linked immunosorbent assay in 15 patients with osteosarcoma before commencing treatment. The patients were divided into two groups, with a high or a low serum VEGF level, and the incidence of metastases and overall survival rate were compared. No significant relationship was observed between the serum VEGF levels and gender, age, the size of the tumour or the response to pre-operative chemotherapy. Patients with a serum VEGF > 1000 pg/ml had significantly worse survival than those with a level < 1000 pg/ml (p = 0.002). The serum VEGF level may be useful in predicting the prognosis for survival in patients with osteosarcoma

Introduction and Aims: To establish whether basic fibroblast growth factor (bFGF) plays a role in the changes in chondrocyte metabolism exhibited in human osteoarthritis (OA). Method: BFGF and its receptor was localised by immunohistochemistry within human OA. The results from OA tissue graded ‘early’ and ‘advanced’ were compared. This was correlated with the identification of proliferating chondrocytes (using by localising PCNA) and dead/dying chondrocytes (using the TUNEL technique). Results: Results showed that bFGF and its receptor were strongly localised around chondrocytes in proliferating clusters in ‘early’ OA, whereas no bFGF was detected in ‘advanced’ OA. In addition, a loss of bFGF activity in ‘advanced’ OA correlated with the identification of large numbers of dead/dying chondrocytes. Conclusion: Results suggest that high levels of bFGF activity in OA play an important role in chondrocyte proliferation and the formation of chondrocyte clusters. In addition, the loss of this activity appears to be directly related to an increase in cell death in ‘advanced’ OA, suggesting that bFGF acts as a ‘survival’ factor in this tissue. The more we understand about the metabolic changes in chondrocytes during OA, the closer we come to delaying or preventing this debilitating joint disease

Introduction: Vascular Endothelial Growth Factor (VEGF) is a proangiogenic cytokine that is expressed highly by many solid tumours often correlating with poor prognosis. VEGF has also been shown to interact with osteoclasts and their precursors in organ cultures to increase differentiation and survival and VEGF receptors have been found on osteoclasts in vitro. In this work we aimed to investigate the expression of VEGF and its receptors in bone metastases from primary breast tumours and further characterise its effects on osteoclasts. We performed immunolocalisation of VEGF in bone metastases and using VEGF and VEGF receptor-specific ligands we assessed their role in osteoclastogenesis in vitro. Methods: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained with antibodies to VEGF and its receptors VEGFR1 and 2, and the macrophage marker CD68. To investigate osteoclastogenesis in vitro Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured over a two-week period under stimulation by cytokines (RANKL, M-CSF, VEGF, PlGF, a specific ligand for VEGFR 1 and VEGF-D, a specific ligand for VEGFR 2). RAW 264.7 cells (a mouse monocyte/macrophage cell line able to differentiate into osteoclast-like cells) were cultured for seven days under stimulation by cytokines (RANKL, VEGF and M-CSF). Osteoclasts were identified by staining for Tartrate Resistant Acid Phophatase (TRAP) and numbers of multinucleated cells counted per treatment. Culture on ivory slices was performed to measure resorption activity of the osteoclasts. Results: The immunohistochemistry demonstrated that breast cancer metastases express VEGF strongly and that the osteoclasts surrounding metastases express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens). The PBMCs stimulated by VEGF and RANKL together differentiated into multinucleated TRAP positive cells in similar numbers (22±4.7) per field of view to the M-CSF and RANKL (27.3±7.2). Resorption of ivory was identified in these cultures. Stimulation with PlGF and RANKL resulted in increased osteoclastogenesis but VEGF-D with RANKL had little effect. Similar results were seen in triplicate experiments RAW 264.7 cells also differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL. Discussion and Conclusions: VEGF is able to induce the differentiation of human and mouse osteoclast-like cells from monocyte precursors in the presence of RANKL and this seems to be mediated by VEGFR1. This may lead to an increase in bone resorption in physiological and pathological situations where there is an increase in VEGF, such as in tumours, embryogenesis and fracture repair. VEGF signalling could be a therapeutic target for osteoclast inhibtion in these situations

The re-establishment of vascularity is an early event in fracture healing; upregulation of angiogenesis may therefore promote the formation of bone. We have investigated the capacity of vascular endothelial growth factor (VEGF) to stimulate the formation of bone in an experimental atrophic nonunion model. Three groups of eight rabbits underwent a standard nonunion operation. This was followed by interfragmentary deposition of 100 μg VEGF, carrier alone or autograft. After seven weeks, torsional failure tests and callus size confirmed that VEGF-treated osteotomies had united whereas the carrier-treated osteotomies failed to unite. The biomechanical properties of the groups treated with VEGF and autograft were identical. There was no difference in bone blood flow. We considered that VEGF stimulated the formation of competent bone in an environment deprived of its normal vascularisation and osteoprogenitor cell supply. It could be used to enhance the healing of fractures predisposed to nonunion

Our aim was to investigate vascular endothelial growth factor (VEGF) expression after lacerations of a meniscus in a rabbit model. Specimens of meniscus were examined using immunohistochemistry, enzyme-linked immunoassay and the reverse transcription polymerase chain reaction after one, two, five or ten weeks. In the periphery of the meniscus 90% of the lacerations had healed after five and ten weeks, but no healing was observed in the avascular area. Expression of VEGF protein and VEGF mRNA was found in the meniscus of both the operated and the contralateral sites but both were absent in control rabbits which had not undergone operation. The highest expression of VEGF was found in the avascular area after one week (p < 0.001). It then lessened at both the vascular and avascular areas, but still remained greater in comparison with the control meniscus (p < 0.05). Despite greater expression of VEGF, angiogenesis failed at the inner portion. These findings demonstrated the poor healing response in the avascular area which may not be caused by an intrinsic cellular insufficiency to stimulate angiogenesis

To determine whether systemic nitric oxide production in tourniquet-induced skeletal muscle ischaemia-reper-fusion injury (SMRI) is dependent on release of vascular endothelial growth factor (VEGF), a modulator of nitric oxide cytoprotection in myocardial ischaemia-reperfusion injury. Mice were randomised (n=10 per group) into: time controls (no tourniquet) and test animals (bilateral hindlimb tourniquet ischaemia). Blood samples were collected in test animals prior to ischaemia and after reper-fusion. In controls, blood samples were collected at the same corresponding time points. Serum VEGF, nitric oxide metabolites (nitrite and nitrate) and the proinflammatory cytokine tumour necrosis factor (TNF)-α (an indicator of systemic inflammation) were determined. At the end of reperfusion, the lungs and muscle (right gastrocnemius) were harvested and tissue injury determined by measuring myeloperoxidase (MPO) activity, a marker of neutrophil infiltration. Data are presented as mean ± SEM and statistical comparison was performed using one-way analysis of variance (ANOVA) with significance attributed to P < 0.05. In comparison to control animals, muscle (4.9±0.3 versus 4±0.03 units/g of wet tissue; P=0.02) and lung (16.7±1.9 versus 10.4±0.5; P=0.005) MPO activity at the end of repercussion was significantly greater in test animals. The table shows the results with respect to serum cytokine levels and nitricxide metabolites. These data demonstrate that SMRI results in local and systemic proinflammatory responses. In contrast to myocardial ischaemia-reperfusion injury, nitric oxide production in tourniquet-induced SMRI is VEGF-independent. Alternative mechanisms for nitric oxide production in tourniquet-controlled extremity surgery requires further evaluation

We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts. Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF. The uptake of [. 3. H]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity

Introduction: Limb reperfusion in patients following pneumatic tourniquet-controlled surgery is associated with nitric oxide (NO) generation. Meanwhile, NO mediates vascular endothelial growth factor (VEGF)-cytoprotection in myocardial ischaemia-reperfusion injury. In addition, VEGF is contributory in attenuating skeletal muscle ischaemia-reperfusion injury (SMRI). Whether this effect of VEGF is NO-mediated in SMRI is unknown. We investigate whether systemic nitric oxide production in tourniquet-induced SMRI is dependent on VEGF release. Methods: Anaesthetised male C57BL/6 mice were randomised (n=10 per group) into two groups: time controls (no tourniquet) and test animals with bilateral hindlimb tourniquets (SMRI; 2 hours of ischaemia, 2 hours of reperfusion). Blood samples were collected in test animals prior to ischaemia and after 2 hours of reperfusion. In controls, blood samples were collected at the same corresponding time points. Serum VEGF, nitric oxide metabolites (nitrite and nitrate) and the proinflammatory cytokine tumour necrosis fractor (TNF)-α (an indicator of systemic inflammation) were determined. At the end of reperfusion, the lungs and muscle (right gastrocnemius) were harvested and tissue injury determined by measuring myeloperoxidase (MPO) activity, a marker of neutrophil infiltration. Data are presented as mean ± SEM and statistical comparison was performed using one-way analysis of variance (ANOVA) with significance attributed to P,0.05. Results: In comparison to control animals, both the muscle (4.9±0.3 versus 4±0.03 units/g of wet tissue; P=0.02) and lung (16.7±1.9 versus 10.4±0.5; P=0.005) MPO activity at the end of reperfusion was significantly greater in test animals. Conclusions: Our data demonstrates that SMRI results in local and systemic proinflammatory responses. In contrast to myocardial ischaemia-reperfusion injury, nitric oxide production in tourniquet-induced SMRI is VEGF-independent. Alternative mechanisms for nitric oxide production in tourniquet-controlled limb surgery requires further evaluation

The authors present case histories relating to experience gained over 18 months of using deantigenic osseous grafts together with platelet growth factor for posterior spine arthrodesis. The spinal column was the last section of the skeleton to recover from the use of this kind of bone graft to promote spinal fusion. This is due to the negative experience encountered during the proposals bought forward during the 1960s and 1970s by French orthopaedic surgeons who used heterologous lyophilised bone for long vertebral arthrodesis. The high incidence of pseudoarthrosis developed as a result of difficulties in absorption of the skeletal implant, which, in order to ensure sufficient mechanical resistance, should have been thicker (8 mm). Therefore it could not be assimilated. This then resulted in relying only on external protection (plastered brace) to provide the necessary rigidity during the process of fusion. The beginning of stable synthesis obtained with transpeduncular fixation and double instrumentation as used in the Cotrel Doubousset technique and methods derived from it made it possible to maintain primary stability over time, therefore positively influencing the process of consolidation. This is why the practice of exclusively using osseous derivations has grown only in the past few years in vertebral surgery, preferring to implant small chips which can be assimilated much easier and to avoid having to search for mechanically resistant areas for bone replacement. In conclusion, the use of heterologous bone grafts in the vertebral column represents a situation similar to that in other mobile osseous areas, in which the immobilisation depends on the use of a mechanical instrument (when it concerns short arthrodesis 2–3 segments). In the case of long spinal fusion such as in correcting vertebral deformity (scoliosis and kyphosis), the problem is more complicated, even though today’s new biological tools (large extension of the bed of arthrodesis) make this possible and offer the advantages of the lack of problems at the donor site. In these cases it is particularly suitable to routinely combine heterologous bone replacement with growth factors (osteoinductive bone proteins) for treatment, which according to our preliminary results should notably shorten the period of assimilation and the fusion healing

Summary Statement. A resorbable and biocompatible polymer-based scaffold was used for the proliferation and delivery of adipose derived stromal cells, as well as delivery of a cell growth/differentiation promoting factor for improved tendon defect regeneration. Introduction. Surgeons perform thousands of direct tendon repairs annually. Repaired tendons fail to return to normal function following injury, and thus require continued efforts to improve patient outcomes. The ability to produce regenerate tendon tissue with properties equal to pre-injured tendon could lead to improved treatment outcomes. The aim of this study was to investigate in vivo tendon regeneration using a biodegradable polymer for the delivery of adipose derived stromal cells (ADSCs) and a polypeptide, growth/differentiation factor-5/(GDF-5), in a tendon gap model. Patients & Methods. Female Fischer 344 rats underwent unilateral Achilles tenotomies. Defects were left un-repaired (Group 1-control), bridged using electrospun 65:35 polylactide-co-glycolide (PLAGA) tubular scaffolds (Group 2), PLAGA/ADSCs (Group 3), or PLAGA/GDF-5 (Group 4) scaffold composites. The plantaris was left intact. Operative limbs were immobilised for 10–14 days, followed by unrestricted activity. The rats were sacrificed at 4 weeks or 8 weeks after surgery, and tendons were assessed with histological, biochemical, and mechanical analyses. Results. PLAGA, PLAGA/ADSCs, and PLAGA/GDF-5 groups showed increased collagen I gene expression at both the 4 and 8 week time points (p<0.05). Tenomodulin (Tnmd) is the mature tendon phenotype marker unique to tendon tissue. Both the PLAGA/ADSCs and PLAGA/GDF-5 groups demonstrated increased tenomodulin expression at 4 and 8 weeks (p<0.05). Ultimate tensile load strength was improved in all PLAGA groups (2, 3, and 4) versus the control. Both composite groups (2 and 3) showed improved collagen deposition, as indicated by increased Collagen Area Fraction (CAF), approaching that of normal tendon at 8 weeks (p<0.05). Scaffold resorption was evident at 4 weeks, with complete replacement of the polymer with regenerate tissue and minimal gap formation at 8 weeks without evidence of an adverse inflammatory reaction. Defects bridged using the scaffold seeded with ADSCs showed improved collagen organization and increased modulus of elasticity compared with controls as well as properties approaching those of native tendon. Discussion/Conclusions. These results demonstrate that a tubular bioresorbable scaffold can promote extracellular matrix synthesis and organization, and the formation of neo-tendinous tissue; as well as serve as a carrier of adipose stromal cells and growth factors that are effective for tendon regeneration. Cells, growth factors and synthetic biomaterial polymers may be combined as a paradigm for regenerative engineering thereby serving as promising options for improved treatments of tendon injuries and potentially improving patient outcomes

Purpose: Apoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Vertebral osteoporotic insufficiency fractures are characterised by pathological rates of osteoblast apoptosis. Skeletal injury in humans results in ‘angiogenic’ responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human vertebral osteoblasts (PHVO) in-vitro. Method: PHVO were cultured from biopsies taken at time of therapeutic vertebroplasty and were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examine their effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined. Results: PHVO expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3ug/mL of mAB VEGF resulted in approx 40% reductions in both. PlGF 50ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 versus 3%). BCl2 transfection gave a 0.9% apoptotic rate. VEGF 10 ng/mL increased BCl2 expression four fold while mAB VEGF decreased it by over 50%. Conclusion: VEGF is a potent regulator of osteoblast life-span in-vitro. This autocrine feedback regulates survival of these cells, mediated via the KDR receptor and expression of BCl2 antiapoptotic gene. This mechanism may represent a novel therapeutic model for the treatment of osteoporosis

Animal studies examining tendon-bone healing have demonstrated that the overall structure, composition, and organization of direct type entheses are not regenerated following repair. We examined the effect of Low-Intensity Pulsed Ultrasound (LIPUS) on tendon-bone healing. LIPUS may accelerate and augment the tendon-bone healing process through alteration of critical molecular expressions. Eight skeletally mature wethers, randomly allocated to either control group (n=4) or LIPUS group (n=4), underwent rotator cuff surgery following injury to the infraspinatus tendon. All animals were sacrificed 28 days post surgery to allow examination of early effects of LIPUS. Humeral head – infraspinatus tendon constructs were harvested and processed for histology and immunohistochemical staining for BMP2, Smad4, VEGF and RUNX2. All the growth factors were semiquantitative evaluated. T-tests were used to examine differences which were considered significant at p < 0.05. Levene's Test (p < 0.05) was used to confirm variance homogeneity of the populations. The surgery and LIPUS treatment were well tolerated by all animals. Placement of LIPUS sensor did not unsettle the animals. Histologic appearance at the tendon-bone interface in LIPUS treated group demonstrated general improvement in appearance compared to controls. Generally a thicker region of newly formed woven bone, morphologically resembling trabecular bone, was noted at the tendon-bone interface in the LIPUS-treated group compared to the controls. Structurally, treatment group also showed evidence of a mature interface between tendon and bone as indicated by alignment of collagen fibres as visualized under polarized light. Immunohistochemistry revealed an increase in the protein expression patterns of VEGF (p = 0.038), RUNX2 (p = 0.02) and Smad4 (p = 0.05) in the treatment group. There was no statistical difference found in the expression patterns of BMP2. VEGF was positively stained within osteoblasts in newly formed bone, endothelial cells and some fibroblasts at the interface and focally within fibroblasts around the newly formed vessels. Expression patterns of RUNX2 were similar to that of BMP-2; the staining was noted in active fibroblasts found at the interface as well as in osteoblast-like cells and osteoprogenitor cells. Immunostaining of Smad4 was present in all cell types at the healing interface. The results of this study indicate that LIPUS may aid in tendon to bone healing process in patients who have undergone rotator cuff repair. This treatment may also be beneficial following other types of reconstructive surgeries involving the tendon-bone interface

Purpose: A preliminary biomechanical test conducted on cadaver specimens validated a new technique for vertebral bone harvesting for anterior intervertebral grafting of the lumbar spine. A cylinder of autologous bone harvested from a neighboring vertebra was used for the intervertebralimplant. The harvesting site was filled with a bone substitute. The biomechanical tests confirmed good restoration of the vertebral body structure. An in vivo study was conducted in the baboon. A block of tricalcium-phosphate (beta-TCP) impregnated with transforming growth factor beta3 (TGF-beta3) was used to fill the bone gap. The purpose of the present study was: 1) to assess the efficacy of this in vivo technique on a primate model, 2) to validate the surgical technique. Material and methods: The retroperitoneal approach was used to operate nine baboons. Eighteen bone cylinders were harvested. The harvesting hole was left empty or filled with a 15 mm diameter beta-TCP cylinder, or with a beta-TCP cylinder impregnated with TGR-beta3. Control scans were obtained at three and six months postoperatively. The baboons were sacrificed at 6 months and the vertebral bodies were removed for histology study. Results: There was no evidence of fracture or loss of vertebral body height. The harvesting holes left empty did not fill, while osteointegration and substantial resorption of the bone substitute was observed in the two other groups. In the group with beta-TCP impregnated with TGF-beta3 the resorption of the cylinder was more complete and signs of prevertebral neoformation of subperiosteal bone, not observed in the beta-TCP group, was observed. There was no neoformation of bone in the spinal canal or in the foramens. The scans showed progressive resorption starting three months postoperatively in the beta-TCP group impregnated with TGF-beta3. Conclusion: The beta-TCP block used is a very good bone substitute for the primate spine. TGF-beta3 accelerates bone resorption and induces neoformation of subperiosteal bone. The new surgical technique for local harvesting of vertebral bone was validated

The immunosuppressive drug rapamycin (RAPA) prevents rejection in organ transplantation by inhibiting interleukin-2-stimulated T-cell division. RAPA has also been suggested to possess strong anti-angiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF). Because VEGF is a key growth factor in fracture healing, the present study was conducted to analyze the effect of RAPA on bone repair. For the herein introduced study 35 SKH-1Hr mice were treated by a daily intraperitoneal (i.p.) injection of RAPA (1.5mg/kg/d) from the day of fracture until sacrifice. Two or five weeks after fracture, animals were killed and bone healing was analyzed using radiological (n=16 at 2 weeks; n=16 at 5 weeks), biomechanical (n=2x8), and histomorphometric (n=2x8). Methods: At 2 weeks additional animals were studied to achieve tissue for protein biochemical analysis of VEGF and proliferating cell nuclear antigen (PCNA; n=3). Additional 34 mice, which received the vehicle only, served as controls. Analyses in controls were similar to those of RAPA-treated animals. X-ray analyses demonstrated that RAPA treatment inhibits callus formation after 2 weeks of fracture healing. The radiologically observed lack of callus formation after RAPA treatment was confirmed by histomorphometric analyses, which revealed a significantly diminished callus size and a reduced amount of bone formation when compared to vehicle-treated controls. Biomechanical testing further demonstrated that RAPA significantly reduces torsional stiffness of the callus (11.5±5.9% of the contralateral unfractured femur vs. 28.3±13.9% in controls; p< 0.05). Of interest, this was associated with a decrease of callus VEGF and PCNA expression. After 5 weeks of fracture healing, however, the negative impact of RAPA on fracture healing was found blunted and the radiological, histomorphometric and biomechanical differences observed after 2 weeks could not longer be detected. We demonstrate that RAPA treatment leads to a severe alteration of early fracture healing. The negative action of RAPA on fracture repair at 2 weeks is most probably due to an inhibition of VEGF expression within the callus as suggested by the results of the Western blot analysis, demonstrating during the early phase of fracture healing a significantly reduced expression of VEGF and PCNA after RAPA treatment. This indicates a substantial alteration of cell proliferation and angiogenic vascularization during initial fracture healing. Since T-cells contribute to delayed fracture healing, RAPA may promote bone healing at later stages due to a reduction of interleukin-2-stimulated Tcell division

Growth factors are reported to play an important role in healing after acute Achilles tendon rupture (ATR). However, the association between growth factors and patient outcome has not been investigated previously. The aim of this retrospective study is to identify growth factors and related proteins which can be used as predictors of healing after ATR, ethical approval was obtained from the Regional Ethical Review Committees in Sweden and followed the guidelines of the Declaration of Helsinki. The study included 28 surgically treated patients (mean age 39.11 ± 8.38 yrs) with acute ATR. Healing was assessed by microdialysate two weeks after the surgery and performed on both injured and contralateral un-injured leg. The microdialysates were analyzed by proteomics based on mass spectrometry (MS) to detect growth factor expressions in ATR patients. One year after the surgery, healing outcomes were evaluated by patient-reported Achilles tendon Total Rupture Score (ATRS), Foot and Ankle Outcome Score (FAOS), and functional outcomes by heel-rise test. A total of 1549 proteins were detected in the microdialysates of which 20 growth factor/ related proteins were identified. 7 of these were significantly up-regulated (IGFBP2, Fold change (FC) = 4.07, P = 0.0036; IGFBP4, FC = 3.06, P = 0.009; CTGF, FC = 15.83, P = 0.003; HDGF, FC = 4.58, P = 0.003; GRB2, FC = 14.8, P = 0.0004; LTBP1, FC = 12.08, P = 0.0008; TGFBI, FC = 5.54, P = 0.001) and 1 down-regulated (IGFBP6) in the injured compared to the contralateral healthy side. Linear regression analysis revealed that TGFB1 was positively associated with improved ATRS (r = 0.585, P = 0.04) as well to ATRS subscales: less limitation in running (r = 0.72, P = 0.004), less jumping limitation (r = 0.764, P = 0.001) and less limitation caused by decreased tendon strength (r = 0.665, P = 0.012). Interestingly, all 7 up-regulated proteins were positively associated with less jumping limitations (IGFBP2, r = 0.667, P = 0.015; IGFBP4, r = 0.675, P = 0.013; CTGF, r = 0.668, P = 0.015; HDGF, r = 0.672, P = 0.014; GRB2, r = 0.665, P = 0.016; LTBP1, r = 0.663, P = 0,016). No associations were observed among any of the growth factor and FAOS or patient's functional outcomes. We conclude that growth factors and related proteins play a crucial role in ATR healing. More specifically, TGFB1 may be used as prognostic biomarker of the patient-reported outcome 1-year post-surgery. These results may be used to develop more specific treatments to improve ATR healing

Tendon and ligament injuries represent highly prevalent and unmet clinical challenge that may significantly benefit from tissue engineering therapeutic strategies, once optimal cell source and biomolecules regulating tendon homeostasis are properly defined. Herein, we aimed to evaluate the expression of tendon/ligament markers in two novel cell populations, namely human dental pulp stem cells (DPSCs) and periodontal ligament cells (PDLCs), in response to supplementation with TGF-β ligands relevant for tendon development and healing, as well as under standard tri-lineage differentiation conditions. DPSCs and PDLCs were isolated from sound human permanent molars removed for orthodontic reasons. Pulp tissue and periodontal ligament were minced and digested with collagenase (3mg/mL) and cells were expanded in α-MEM supplemented with 10% fetal bovine serum (basal medium). To evaluate the susceptibility of DPSCs and PDLCs to tenogenic induction, cells were seeded at density of 1000 cells/cm² and cultured up to 21 days in basal medium or media supplemented with TGF-β3 (10ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated weakly by analysis of expression of tendon, bone and cartilage markers, employing real time RT-PCR and immunocytochemistry. A significant increase in collagen I and collagen III expression was observed with the culture progression in all conditions, with abundant matrix being deposited by day 14. A significant upregulation of scleraxis expression was demonstrated in response to supplementation with TGF-β3 in both cell populations, when compared to basal medium and medium with GDF-5. It was concluded that TGF-β3 may represent an effective inducer of stem cell tenogenic differentiation.

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

Study Design: Prospective randomised controlled trial. Objective: To determine whether topical application of autologous growth factors (AGF) and thrombin as a spray to large spinal wounds can reduce blood loss after lumbar spinal fusion. Subjects: Seventy patients undergoing single or multi-level lumbar spinal fusion were randomised to receive a spray of AGF and thrombin to the raw surfaces of the wound as a haemostat or to receive no spray, immediately prior to wound closure. Outcome Measures: Intra-operative swab and suction blood loss and post-operative loss into suction drains. Results: The median intra-operative blood loss in the two groups was similar (665 ml in the AGF/Thrombin spray group and 500 ml in the non-spray group), but the post-operative loss in the AGF/Thrombin spray group was 34% less than in the controls when expressed as the ratio of intra- to post-operative blood loss (median postoperative loss in AGF / Thrombin spray group 315 ml; median loss in the non-spray group 360 ml). There was an even greater difference (44%) when intra-operative blood loss was more than 500ml (median loss in AGF/Thrombin spray group 340 ml; median loss in the non-spray group 520 ml). Conclusions: AGF/Thrombin spray applied to lumbar spine wounds reduces blood loss generally, but more particularly when the intra-operative loss has been considerable. This effect is probably due to local replacement of haemostatic agents when these have been systemically depleted by the surgical procedure. This study provides evidence to support the use of AGF/Thrombin spray as an effective haemostatic agent in major spinal surgery

Our study sets out to show whether vascular endothelial growth factor (VEGF) expression in stage 2B osteosarcomas around the knee influences disease-free and overall survival. Fifty-two such patients treated in out unit were identified and followed-up for for a minimum of 92 months. All were treated according to the current MRC protocol and had resection of their tumour. Tissue from their resected tumours was stained for VEGF using immunohistochemical methods and the percentage of tumour cells staining for VEGF was assessed. The relationship between VEGF expression and survival was assessed using the log-rank test and Kaplan-Meier survival curves. At follow-up 32 (62%) patients were dead, all from metastatic disease. Twenty-six (50%) tumours showed expression of VEGF. Statistical analysis showed that patients with tumours with VEGF expression in more than 25% of the cells had significantly shorter overall survival (p=0.019) and disease free intervals (p=0.009). VEGF is peptide which acts as a stimulator of new blood vessel growth in normal tissues, as well as in some solid tumours and their metastases. A tumour which is able to induce a blood supply has an increased ability to grow, seed metastases and threaten life. Our study is the first to look at VEGF expression in the tumour cells surviving after chemotherapy. It is this population of cells which is important as it is these cells which may go on to develop into metastatic or locally recurrent tumours. The over-expression of VEGF by osteosarcoma cells is thought to be associated with a worse prognosis due to a number of mechanisms. This study shows that VEGF expression is an important prognostic factor in osteosarcomas. Suppression of tumour angiogenesis by inhibition of the action of VEGF has shown promise in animal models as a potential new treatment for osteosarcoma, and warrants further study

Our study sets out to show whether vascular endothelial growth factor (VEGF) expression in stage 2B osteosarcomas around the knee influences disease-free and overall survival. Fifty-two such patients treated in out unit were identified and followed-up for for a minimum of 92 months. All were treated according to the current MRC protocol and had resection of their tumour. Tissue from their resected tumours was stained for VEGF using immunohistochemical methods and the percentage of tumour cells staining for VEGF was assessed. The relationship between VEGF expression and survival was assessed using the log-rank test and Kaplan-Meier survival curves. At follow-up 32 (62%) patients were dead, all from metastatic disease. Twenty-six (50%) tumours showed expression of VEGF. Statistical analysis showed that patients with tumours with VEGF expression in more than 25% of the cells had significantly shorter overall survival (p=0.019) and disease free intervals (p=0.009). Expression of VEGF also correlated with expression of the proteolytic enzyme MMP9 (p=0.02). VEGF is peptide which acts as a stimulator of new blood vessel growth in normal tissues, as well as in some solid tumours and their metastases. A tumour which is able to induce a blood supply has an increased ability to grow, seed metastases and threaten life. Our study is the first to look at VEGF expression in the tumour cells surviving after chemotherapy. It is this population of cells which is important as it is these cells which may go on to develop into metastatic or locally recurrent tumours. The over-expression of VEGF by osteosarcoma cells is thought to be associated with a worse prognosis due to a number of mechanisms. This study shows that VEGF expression is an important prognostic factor in osteosarcomas and suggests that the mechanisms by which VEGF and MMP9 expression produce a poor prognosis may be linked. Suppression of tumour angiogenesis by inhibition of the action of VEGF has shown promise in animal models as a potential new treatment for osteosarcoma, and warrants further study

Background Apoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Skeletal injury in humans results in angiogenic responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human osteoblasts(PHOB) in vitro. Methods PHOB were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examine their effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis.. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined. Results PHOB expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3ug/mL of mAB VEGF resulted in approx 40% reductions in both. PlGF 50ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 vs 3%). BCl2 transfection gave a 0.9% apoptotic rate. VEGF 10 ng/mL increased BCl2 expression 4 fold while mAB VEGF decreased it by over 50%. Conclusions VEGF is a potent regulator of osteoblast lifespan in vitro. This autocrine feedback regulates survival of these cells, mediated via the KDR receptor and expression of BCl2 antiapoptotic gene

Introduction. Differing levels of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiology of tendon degeneration and retraction is unclear. Neoangiogenesis in tendon parenchyma indicates degeneration. Hypoxia inducible factor 1(HIF) and vascular endothelial growth factor (VEGF) are important inducers of neoangiogenesis. Rotator cuff tendons rupture leads to fatty muscle infiltration (FI) and muscle atrophy (MA). The aim of this study is to clarify the relationship between HIF and VEGF expression, neoangiogenesis, FI, and MA in tendon retraction found in full-thickness rotator cuff tears. Methods. Rotator cuff tendon samples of 33 patients with full-thickness medium-sized rotator cuff tears were harvested during reconstructive surgery. The samples were dehydrated and paraffin embedded. For immunohistological determination of VEGF and HIF expression, sample slices were strained with VEGF and HIF antibody dilution. Vessel density and vessel size were determined after Masson-Goldner staining of sample slices. The extent of tendon retraction was determined intraoperatively according to Patte's classification. Patients were assigned to 4 categories based upon Patte tendon retraction grade, including one control group. FI and MA were measured on standardized preoperative shoulder MRI. Results. HIF and VEGF expression, FI, and MA were significantly higher in torn cuff samples compared with healthy tissue (p<0.05). HIF and VEGF expression, and vessel density significantly increased with extent of tendon retraction (p<0.04). A correlation between HIF/VEGF expression and FI and MA could be found (p<0.04). There was no significant correlation between HIF/VEGF expression and neovascularity (p>0.05). Conclusion. Tendon retraction in full-thickness medium-sized rotator cuff tears is characterized by neovascularity, increased VEGF/HIF expression, FI, and MA. VEGF expression and neovascularity may be effective monitoring tools to assess tendon degeneration

Purpose: To determine if administration of recombinant bFGF in an alginate gel would increase early healing mechanical parameters in acutely injured rat rotator cuff tendon at specific time points. Method: Sprague Dawley rats were randomly divided into 2 groups and had surgically created 1mm (half tendon width) full thickness injuries at exactly 2mm from insertion site of Infraspinatus on the humerus. 200ng of bFGF or vehicle control was administered to randomly chosen rats. Tendons were harvested at 1 week, 2 weeks and 4 weeks. In both groups, the Infraspinatus tendon was dissected, and left attached to the humerus. At the time of testing, the intact portion of the injured tendon was divided sharply across tendon fibers at the level of the injury leaving only the healing tissue callus in continuity with the remaining proximal and distal portions of the tendon and loaded to failure. Results: At 1 week the injury group’s average load to failure was 0.60N versus 0.61N in the bFGF injury group P = 1.000. At 2 weeks the injury group’s average load to failure increased to 1.03N versus 2.08N in the bFGF injury group P = 0.440 At 4 weeks the injury group’s average load to failure increased to 3.93N versus 5.56N in the bFGF injury group P = 0.008 representing a 41% increase in ultimate load. At 4 weeks, callus size of the injury group was 0.4mm2 versus 2.7mm2 in the bFGF injury group P < 0.001. Stiffness at 4 weeks for the injury tendons was 2.15 N/mm versus 3.54 N/mm in the bFGF group P = 0.008. Conclusion: At 4 weeks healing tissue of acutely injured rotator cuff exposed to bFGF has an increase in ultimate load to failure (41% compared to control), increase in tendon callus size and stiffness. Our findings suggest a role of bFGF or similar growth factors in accelerating the healing of injured rotator cuff tendon

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230