Insulin‐like growth factor‐I improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells

Human umbilical cord mesenchymal stromal cells (hUCMSCs) are an attractive cell source for tissue engineering with numerous advantages over other adult stem cell sources, such as great expansion ability in vitro and extensive availability. The objective of this 6‐week study was to test the hypothesis that switching from chondrogenic transforming growth factor‐beta3 (TGF‐β3) to anabolic insulin‐like growth factor‐I (IGF‐I) at the 3‐week time point would produce more cartilage‐like matrix than TGF‐β3 alone. hUCMSCs were seeded into polyglycolic acid (PGA) scaffolds and then cultured in chondrogenic medium containing TGF‐β3 for 3 weeks. The TGF‐β3‐treated hUCMSCs were then exposed for 3 more weeks to one of four different conditions: (1) continued in chondrogenic medium, (2) control medium (no TGF‐β3), (3) control medium with 10 ng/ml IGF‐I, or (4) control medium with 100 ng/ml IGF‐I. Compared to continuing with TGF‐β3, switching to IGF‐I increased collagen production, and furthermore increased both collagen type II gene expression and immunostaining. In conclusion, the shift from TGF‐β3 to IGF‐I at week 3 resulted in a significant increase of cartilage‐like extracellular matrix, confirming our hypothesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1109–1115, 2009     

Temporal growth factor release from platelet‐rich plasma,trehalose lyophilized platelets,and bone marrow aspirate and their effect on tendon and ligament gene expression

Platelet‐rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet α‐granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF‐β1 and PDGF‐BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT‐PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases‐3 and 13 (MMP‐3, MMP‐13) was performed. TGF‐β1 and PDGF‐BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF‐β1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP‐13 expression. BMA resulted in decreased COMP and increased MMP‐3 and MMP‐13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP‐13, and MMP‐3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1033–1042, 2009     

Relationship of cytokine levels and clinical effect on platelet‐rich plasma‐treated lateral epicondylitis

Lateral epicondylitis (LE) is difficult to manage and can result in significant patient morbidity. Currently, the clinical use of platelet‐rich plasma (PRP) for painful tendons has received attention, but its efficacy remains controversial. This study aimed to investigate the clinical effects of PRP and its biological components. A total of 156 patients with LE were randomly divided into group 1, treated with a single injection of 2‐ml autologous PRP, and group 2, treated with a control received only physical therapy without injection. Both groups used a tennis elbow strap and performed stretching and strengthening exercises during 24 weeks’ follow‐up. Pain and functional improvements were assessed using the visual analog scale (VAS), Modified Mayo Clinic Performance Index for the elbow, and magnetic resonance imaging (MRI). White blood cell count, platelet count, and levels of platelet‐derived growth factor‐AB (PDGF‐AB), PDGF‐BB, transforming growth factor‐β (TGF‐β), vascular endothelial growth factor, epithelial growth factor, and interleukin‐1 β in PRP were measured and investigated for statistical correlation with the clinical score. At 24 weeks, all pain and functional variables, including VAS score, Mayo Clinic performance scores, and MRI grade, improved significantly in group 1 (p < 0.05). PDGF‐AB, PDGF‐BB, and TGF‐β levels were more significantly increased in PRP than in whole blood. TGF‐β level significantly correlated with Mayo Clinic performance score and MRI grade improvement. Thus, TGF‐β level in PRP is considered to play a pivotal role in tendon healing. These results may contribute to identifying the best protocol for PRP application in tendinopathies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:913–920, 2018.

     

Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin

Platelet‐rich fibrin (PRF^®) is an autologous fibrin sealant (FS) enriched with a platelet concentrate (>1,000,000 platelets/μL) produced by the automated Vivostat^® system and used to enhance wound healing. The effects of PRF were compared with supernatant from thrombin‐activated platelet concentrate, recombinant human platelet‐derived growth factor (rhPDGF) isoforms, and a homologous FS in cultured normal human dermal fibroblasts. Also, the release of selected endogenous growth factors from PRF and their stability against proteolytic degradation were studied. The proliferative effect of PRF exceeded that of FS and rhPDGF‐BB, although it was lower than thrombin‐activated platelet concentrate possibly due to sustained growth factor release from platelets in PRF. Anti‐PDGF antibody blocked the mitogenic effect of rhPDGF‐BB but not that of PRF in growth‐arrested fibroblasts. PRF promoted secretion of carboxyterminal propeptide of type I collagen into conditioned medium while rhPDGF‐AB had no significant effect on collagen biosynthesis. Limited proteolysis of PDGF‐AB and no proteolysis of transforming growth factor‐β1 (TGF‐β1) in PRF were observed with trypsin treatment, whereas rhPDGF‐AB and rhTGF‐β1 in bovine serum albumin, matching the total protein concentration of PRF, were almost completely degraded after 24 hours at 37 °C. To conclude, PRF provides sustained release and protection against proteolytic degradation of endogenous fibrogenic factors important for wound healing.     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

Intraarticular injection autologous platelet‐rich plasma and bone marrow concentrate in a goat osteoarthritis model

To evaluate the effects of intraarticular injections of autologous platelet‐rich plasma (PRP) or bone marrow concentrate (BMC) on osteoarthritis (OA), 24 adult goats were equally divided into control (Ctrl), saline (NS), PRP, and BMC groups, and OA was induced by surgery in NS, PRP, and BMC groups. Autologous PRP and BMC were obtained from whole blood and bone marrow aspirates, respectively. The data revealed, platelets were increased in BMC by 1.8‐fold, monocytes by 5.6‐fold, TGF‐β1 by 7.7‐fold, and IGF‐1 by 3.6‐fold (p < 0.05), and platelets were increased in PRP by 2.9‐fold, and TGF‐β1 by 3.3‐fold (p < 0.05). From the sixth week post‐operation, saline, PRP, and BMC were administered by intraarticular injection once every 4 weeks, three consecutive times. After the animals were sacrificed, inflammatory cytokines in the synovial fluid was measured, and bone and cartilage degeneration progression was observed by macroscopy, histology, and immunohistochemistry. Compared with the NS group, the level of inflammatory cytokines was reduced in the PRP and BMC groups (p < 0.05). Histologically, delayed cartilage degeneration and higher levels of extracellular matrix (ECM) were observed in both PRP and BMC treated groups (p < 0.05). Furthermore, the BMC group showed greater cartilage protection and less ECM loss than the PRP group (p < 0.05). In summary, this study showed that intraarticular injection of autologous PRP and BMC has therapeutic efficacy in a goat osteoarthritis model, with the greater benefit in terms of cartilage protection being observed in the BMC‐treated group than PRP. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2140–2146, 2018.

     

Transforming growth factor β1 regulates the expression of CCN2 in human keratinocytes via Smad‐ERK signalling

Connective tissue growth factor (CCN2/CTGF) and transforming growth factor β1 (TGF‐β1) are important regulators of skin wound healing, but controversy remains regarding their expression in epithelial cell lineages. Here, we investigate the expression of CCN2 in keratinocytes during reepithelialisation and its regulation by TGF‐β1. CCN2 was detected in the epidermis of healing full‐thickness porcine wounds. Human keratinocytes were incubated with or without 10 ng/ml TGF‐β1, and signalling pathways were blocked with 10‐μM SIS3 or 20‐μM PD98059. Semi‐quantitative real‐time PCR was used to study CCN2 mRNA expression, and western blot was used to measure CCN2, phosphorylated‐ERK1/2, ERK1/2, phosphorylated‐Smad3 and Smad2/3 proteins. CCN2 was transiently expressed in neoepidermis at the leading edge of the wound in vivo. In vitro, CCN2 expression was induced by TGF‐β1 at 2 hours (7·5 ± 1·9‐fold mRNA increase and 3·0 ± 0·6‐fold protein increase) and 12 hours (5·4 ± 1·9‐fold mRNA increase and 3·3 ± 0·6‐fold protein increase). Compared with inhibiting the SMAD pathway, inhibiting the mitogen‐activated protein kinase (MAPK) pathway was more effective in reducing TGF‐β1‐induced CCN2 mRNA and protein expression. Inhibition of the MAPK pathway had minimal impact on the activity of the SMAD pathway. CCN2 is expressed in keratinocytes in response to tissue injury or TGF‐β1. In addition, TGF‐β1 induces CCN2 expression in keratinocytes through the ras/MEK/ERK pathway. A complete understanding of CCN2 expression in keratinocytes is critical to developing novel therapies for wound healing and cutaneous malignancy.     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011