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

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

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

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

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

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

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

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

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