Release of growth factors from a reinforced collagen GAG matrix supplemented with platelet rich plasma: Influence on cultured human meniscal cells

Damage to meniscal cartilage has been strongly linked to accelerated articular wear and consequently to osteoarthritis. Damage might be ameliorated by delivery of growth factors from platelet rich plasma (PRP) via a fiber reinforced collagen matrix designed for meniscal repair. PRP composition, release of growth factors, and influence on meniscal cell growth and gene expression were investigated. PRP was prepared using Harvest Smartprep (HS‐PRP), Cascade Fibrinet (CF‐PRP), and a simple centrifuge protocol (DC‐PRP) from four donors each. CF‐PRP had the highest ratio of platelets, with very few other blood cell types. HS‐PRP had the highest total number of platelets but also contained high levels of red and white blood cells. Absorbed to collagen matrices HS‐PRP released the highest levels of TGF‐β1 and PDGF‐AB with DC‐PRP the most IGF‐1. Cumulative release from collagen matrix was 48 ng/cm³ IGF‐1, 96 ng/cm³ TGF‐β1, and 9.6 ng/cm³ PDGF‐AB. Collagen matrix with PRP was able to increase meniscal cell number above peripheral whole blood and up‐regulated gene expression of Aggrecan, Collagen type I (α1), and Elastin (3.3 ± 0.8‐fold, 2.9 ± 0.6‐fold, 4.0 ± 1.4‐fold, respectively). Demonstrating that PRP combined with fiber reinforced collagen matrix could influence meniscal cells and might be of use for treating meniscal defects. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:273–278, 2014.     

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet‐derived growth factor‐BB (PDGF‐BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF‐BB, and HBO combined with PDGF‐BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF‐α receptor (PDGFR‐α) and β receptor (PDGFR‐β) was detected by RT‐PCR. Protein expression of PDGFR‐β was detected by Western blotting. In vivo, chondrocytes and PDGF‐BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF‐BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up‐regulated mRNA expression of PDGFR‐α and increased cell accumulation in the S‐phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR‐β than HBO or PDGF‐BB. In vivo results suggested that PDGF‐BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin‐O and type II collagen staining confirmed the hyaline‐like cartilage regeneration in the repaired tissues. In situ up‐regulation of PDGFR‐β expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1439–1446, 2009     

Age dependence of expression of growth factor receptors in porcine ACL fibroblasts

Tissue engineering approaches that harness the stimulatory power of platelet‐rich plasma have produced encouraging results in anterior cruciate ligament (ACL) repair. However, a number of recent studies have demonstrated age‐dependent differences in cellular responses to such an approach. Identifying the reasons for these differences would allow counteracting them and consequently improve outcomes. In this study we hypothesized that these age‐related effects are caused by differences in the expression of the receptors for growth factors released from platelet‐rich plasma (PRP). Porcine ACL fibroblasts from a predetermined number of animals of different ages were obtained, and mRNA levels of the receptors of platelet‐derived growth factor (PDGF), transforming growth factor β (TGF‐β), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) were determined. Expression levels were compared across age groups (young and adolescent) and regressed on age in days. While no significant difference was seen across groups, the regression analysis showed decreases in receptor expression with increasing age. These differences were statistically significant for TGF‐β receptor 1, FGF receptor, and VEGF receptor 2; and borderline significant for TGF‐β receptor 3 and PDGF receptor. The only receptor that was not associated with age was VEGF receptor 1, a regulator of VEGF receptor 2. These findings suggest that the decrease in growth factor receptor expression as a likely reason for reduced PRP action with increasing age. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1107–1112, 2010     

Heparin‐Conjugated Poly(Lactic‐Co‐Glycolic Acid) Nanospheres Enhance Large‐Wound Healing by Delivering Growth Factors in Platelet‐Rich Plasma

Platelet‐rich plasma (PRP) contains many growth factors that are involved in tissue regeneration processes. For successful tissue regeneration, protein growth factors require a delivery vehicle for long‐term and sustained release to a defect site in order to maintain their bioactivity. Previously, we showed that heparin‐conjugated poly(lactic‐co‐glycolic acid) nanospheres (HCPNs) can provide long‐term delivery of growth factors with affinity for heparin. In this study, we hypothesize that treatment of a skin wound with a mixture of PRP and HCPNs would provide long‐term delivery of several growth factors contained in PRP to promote the skin wound healing process with preservation of bioactivity. The release of platelet‐derived growth factor‐BB (PDGF‐BB), contained in PRP, from HCPN with fibrin gel (FG) showed a prolonged release period versus a PRP mixture with FG alone (FG‐PRP). Also, growth factors released from PRP with HCPN and FG showed sustained human dermal fibroblast growth for 12 days. Full‐thickness skin wound treatment in mice with FG‐HCPN‐PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 9 compared with treatment with FG‐HCPN or FG‐PRP. The enhanced wound healing using FG‐HCPN‐PRP may be due to the prolonged release not only of PDGF‐BB but also of other growth factors in the PRP. The delivered growth factors accelerated angiogenesis at the wound site.     

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     

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     

Platelet‐rich plasma (PRP) impairs the craniofacial bone repair associated with its elevated TGF‐β levels and modulates the co‐expression between collagen III and α‐smooth muscle actin

Transforming growth factor‐β (TGF‐β) is considered the main inducer of both the α‐smooth muscle actin (α‐SMA) phenotype and collagen synthesis and deposition and plays a significant role in the tissue repair and the development of fibrosis. Since the PRP constitutes an important source of TGF‐β and its efficacy on the craniofacial bone repair remains controversy, the aim of this study was to evaluate the effect of PRP in the presence of levels of TGF‐β on PRP samples, as well as in the presence of collagen III and α‐SMA+ cells, while comparing these results by means of a histomorphometric analysis of the bone matrix and fibrous deposition on the bone repair. Four bone defects of 16 mm² were created on the calvarium of 21 rabbits. The surgical defects were treated with either particulate autograft, particulate autograft mixed with PRP and PRP alone. Animals were euthanized at 15, 30, and 45 days postoperative. Histomorphometric and immunohistochemical analyses were performed to assess repair time, as well as the expression of collagen III, and α‐SMA. The histomorphometric results demonstrated intensive deposition of fibrous tissue while hinder bone deposition occurred in PRP groups. These results coincided with higher values of the TGF‐β on the PRP sample, also larger occurrence of diffuse collagen III deposition and higher presence of α‐SMA+ cells spread among the fibrous tissue. Thus, the higher levels of TGF‐β associated with the both expression of collagen III and α‐SMA on defect treated with PRP suggest that its biomaterial induce an effect that can be considered similarly to a fibroproliferative disorder. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:457–463, 2011     

Comparison of the effect of intra‐tendon applications of recombinant human platelet‐derived growth factor‐BB,platelet‐rich plasma,steroids in a rat achilles tendon collagenase model

This study compared the effect of intra‐tendon (IT) delivery of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB), platelet‐rich plasma (PRP) and corticosteroids in a rat tendinopathy model. Seven days after collagenase induction of tendinopathy, a 30‐µl IT injection was administered. Treatments included: saline; 3 µg rhPDGF‐BB; 10 µg rhPDGF‐BB; PRP; and 300 µg triamcinolone acetonide (TCA). Outcomes were assessed 7 and 21 days after treatment. All groups exhibited good to excellent repair. Relative to saline, cell proliferation increased 65% in the 10 µg rhPDGF‐BB group and decreased 74% in the TCA group; inflammation decreased 65% in the TCA group. At 7 days, maximum load‐to‐failure was increased in the 3 µg rhPDGF‐BB group relative to saline, PRP, and TCA (p < 0.025). On day 21, maximum load‐to‐rupture was increased in the 10 µg rhPDGF‐BB group relative to saline, PRP, and TCA (p < 0.035) and in the 3 µg rhPDGF‐BB group compared to saline and TCA (p < 0.027). Stiffness in the 10 µg rhPDGF‐BB group was increased compared to saline, PRP, and TCA (p < 0.038). Histology demonstrated similar repair in all groups. PRP and TCA did not improve mechanical properties compared to saline. Injections of rhPDGF‐BB increased maximum load‐to‐failure (3 and 10 µg) and stiffness (10 µg) relative to controls and commonly used treatments. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:145–150, 2014.     

肘关节后外侧旋转不稳定韧带损伤的载线影像学特点研究

目的 ：探讨肘关节外侧韧带复合体对肘关节后外侧旋转不稳定的作用及韧带损伤时肘关节侧位X线影像学中桡肱率变化特点。方法：取冷冻保存的新鲜成人肘关节标本20侧,将标本制成肘关节＂骨-韧带＂标本,随机分为两组。利用自行设计的维持肘关节后外侧旋转活动度的设备固定标本,A组按次序切断外侧结构：A1组,外侧韧带完整的标本组;A2组,切断桡侧尺副韧带组;A3组,增加切断环状韧带组;A4组,最后切断桡侧副韧带组。B组分为：B1组,外侧韧带完整的标本组;B2组,切断桡侧副韧带组;B3组,再切断环状韧带组;B4组,最后切断桡侧尺副韧带组。分别摄肘关节侧位X线片,在PACS系统中测量桡肱率,比较各组的差异,并统计分析。结果：A组,统计分析各组数据,各组间桡肱率增加差异有统计学意义。B组,统计分析前2次切断韧带,各组间桡肱率增加差异无统计学意义。切断桡侧尺副韧带（B4组）,与前3组比较差异有统计学意义。结论：桡侧尺副韧带是限制肘关节后外侧旋转脱位的主要结构,环状韧带、桡侧副韧带为次要结构;并根据肘关节后外侧旋转不稳定X线影像学特点将其分为4个等级。     

自体富含血小板血浆与全血痛点注射治疗网球肘的病例对照研究

目的 ：比较自体富含血小板血浆与全血注射治疗网球肘的临床疗效。方法 ：2011年1月至2014年1月,门诊诊治慢性网球肘患者40例,分成两组,每组20例。一组给予自体富含血小板血浆痛点注射（PRP组）,男5例,女15例;平均年龄（47.50±9.86）岁;平均病程（4.67±3.27）个月。另一组给予自体全血痛点注射（AWB组）,男3例,女17例;平均年龄（46.50±9.96）岁;平均病程（4.53±2.27）个月。注射后给予肘关节弹力绷带制动,指导患者行伸展及力量锻炼。术后即刻及术后4、8周采用疼痛视觉模拟评分（visual analog scale,VAS）、Mayo肘关节评分及压痛阈值（pressure pain threshold,PPT）进行疗效评价。结果 ：全部患者获得随访,未发现相关并发症。VAS、Mayo评分及PPT值,PRP组分别由治疗前7.22±1.32、56.71±10.90和17.47±4.62改善至治疗后8周的2.73±1.00、91.59±6.95和21.35±4.80;AWB组分别由治疗前7.16±1.89、54.72±8.36和17.06±4.83改善至治疗后8周的3.81±1.36、82.06±7.89和20.12±4.97。两组治疗后4周评分均优于治疗前。治疗后4周,两组间PPT比较,差异无统计学意义;VAS、Mayo评分比较,AWB组低于PRP组。治疗后8周,AWB组VAS高于PRP组,Mayo评分及PPT均低于PRP组。结论：自体富含血小板血浆痛点注射治疗网球肘较自体全血痛点注射方法在疼痛缓解和功能改善方面疗效更好且持久。     

Endothelial cells incubated with platelet‐rich plasma express PDGF‐B and ICAM‐1 and induce bone marrow stromal cell migration

Platelet‐rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor‐A (VEGF‐A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor‐B (PDGF‐B), and this effect was not inhibited by an anti‐VEGF‐A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule‐1 (ICAM‐1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor κB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro‐osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro‐osteolytic properties. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1493–1498, 2009     

Recombinant human platelet‐derived growth factor BB (rhPDGF‐BB) and beta‐tricalcium phosphate/collagen matrix enhance fracture healing in a diabetic rat model

Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet‐derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 µg) and high (75 ug) doses of recombinant human PDGF‐BB (rhPDGF‐BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle‐treated animals. rhPDGF‐BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF‐BB treatment significantly increased callus peak torque values (p < 0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF‐BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF‐BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF‐BB may be a new therapeutic approach to treat diabetes‐impaired fracture healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1074–1081, 2009     

Regulation of RANKL‐induced osteoclastogenesis by TGF‐β through molecular interaction between Smad3 and Traf6

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling. © 2011 American Society for Bone and Mineral Research.     

Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c‐Fos in RANKL‐Induced Osteoclastogenesis

We have previously reported that transforming growth factor β (TGF‐β) plays an essential role in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE‐seq and ChIP‐seq) analyses indicated the cooperation of Smad2/3 with c‐Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c‐Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF‐β, and c‐Fos binding to open chromatin sites was suppressed by inhibition of TGF‐β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c‐Fos deficiency. These results suggest that TGF‐β regulates RANKL‐induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c‐Fos. © 2014 American Society for Bone and Mineral Research.     

Enhanced flexor tendon healing through controlled delivery of PDGF‐BB

A fibrin/heparin‐based delivery system was used to provide controlled delivery of platelet derived growth factor BB (PDGF‐BB) in an animal model of intrasynovial flexor tendon repair. We hypothesized that PDGF‐BB, administered in this manner, would stimulate cell proliferation and matrix remodeling, leading to improvements in the sutured tendon's functional and structural properties. Fifty‐six flexor digitorum profundus tendons were injured and repaired in 28 dogs. Three groups were compared: (1) controlled delivery of PDGF‐BB using a fibrin/heparin‐based delivery system; (2) delivery system carrier control; and (3) repair‐ only control. The operated forelimbs were treated with controlled passive motion rehabilitation. The animals were euthanized at 7, 14, and 42 days, at which time the tendons were assessed using histologic (hyaluronic acid content, cellularity, and inflammation), biochemical (total DNA and reducible collagen crosslink levels), and biomechanical (gliding and tensile properties) assays. We found that cell activity (as determined by total DNA, collagen crosslink analyses, and hyaluronic acid content) was accelerated due to PDGF‐BB at 14 days. Proximal interphalangeal joint rotation and tendon excursion (i.e., tendon gliding properties) were significantly higher for the PDGF‐BB‐treated tendons compared to the repair‐alone tendons at 42 days. Improvements in tensile properties were not achieved, possibly due to suboptimal release kinetics or other factors. In conclusion, PDGF‐BB treatment consistently improved the functional but not the structural properties of sutured intrasynovial tendons through 42 days following repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Transforming growth factor‐β (TGF‐β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome

Non‐inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor‐β (TGF‐β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF‐β and connective tissue growth factor (CTGF), a downstream mediator of TGF‐β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF‐β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF‐β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF‐β1 and CTGF, (R² = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF‐β1 (R² = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF‐β and CTGF, a TGF‐β regulated protein, and that this TGF‐β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:116–122, 2014.     

Dullard/Ctdnep1 Regulates Endochondral Ossification via Suppression of TGF‐β Signaling

Transforming growth factor (TGF)‐β signaling plays critical roles during skeletal development and its excessive signaling causes genetic diseases of connective tissues including Marfan syndrome and acromelic dysplasia. However, the mechanisms underlying prevention of excessive TGF‐β signaling in skeletogenesis remain unclear. We previously reported that Dullard/Ctdnep1 encoding a small phosphatase is required for nephron maintenance after birth through suppression of bone morphogenetic protein (BMP) signaling. Unexpectedly, we found that Dullard is involved in suppression of TGF‐β signaling during endochondral ossification. Conditional Dullard‐deficient mice in the limb and sternum mesenchyme by Prx1‐Cre displayed the impaired growth and ossification of skeletal elements leading to postnatal lethality. Dullard was expressed in early cartilage condensations and later in growth plate chondrocytes. The tibia growth plate of newborn Dullard mutant mice showed reduction of the proliferative and hypertrophic chondrocyte layers. The sternum showed deformity of cartilage primordia and delayed hypertrophy. Micromass culture experiments revealed that Dullard deficiency enhanced early cartilage condensation and differentiation, but suppressed mineralized hypertrophic chondrocyte differentiation, which was reversed by treatment with TGF‐β type I receptor kinase blocker LY‐364947. Dullard deficiency induced upregulation of protein levels of both phospho‐Smad2/3 and total Smad2/3 in micromass cultures without increase of Smad2/3 mRNA levels, suggesting that Dullard may affect Smad2/3 protein stability. The phospho‐Smad2/3 level was also upregulated in perichondrium and hypertrophic chondrocytes in Dullard‐deficient embryos. Response to TGF‐β signaling was enhanced in Dullard‐deficient primary chondrocyte cultures at late, but not early, time point. Moreover, perinatal administration of LY‐364947 ameliorated the sternum deformity in vivo. Thus, we identified Dullard as a new negative regulator of TGF‐β signaling in endochondral ossification. © 2014 American Society for Bone and Mineral Research.