Differential gene expression and immunolocalization of insulin‐like growth factors and insulin‐like growth factor binding proteins between experimental nonunions and standard healing fractures

Insulin‐like growth factors (IGF‐I/II) are important growth factors in bone, and their actions are regulated by six IGF binding proteins (IGFBPs). However, little is known about their exact functions in fracture healing. The aim of this study was to compare the gene expression and immunolocalization of IGFs and IGFBPs between standard healing fractures and nonunions using rat experimental models. Standard healing fractures and nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of the healing fractures and the fibrous tissue of the nonunions, and gene expression were analyzed by real‐time PCR. Additionally, immunolocalization of these proteins was studied by immunohistochemistry at postfracture days 7, 14, and 21. In nonunions, the gene expression of IGF‐I/II and IGFBP‐6 was significantly higher, and that of IGFBP‐5 was significantly lower at several time points. The immunolocalization of IGF‐I/II and IGFBP‐5 was widely distributed in both models. In contrast, that of IGFBP‐6 was barely detected in the fracture callus. In conclusion, our results suggest that IGFs/IGFBPs may have important roles not only in fracture healing but also in nonunion formation. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1820–1826, 2011     

Altered relative expression of BMPs and BMP inhibitors in cartilaginous areas of human fractures progressing towards nonunion

The present study was conducted to evaluate the hypothesis that an imbalance in the local production of bone morphogenetic proteins (BMPs) and BMP inhibitors exists within the cartilaginous intermediate of nonhealing fractures. Biopsies were recovered intraoperatively from human fractures that, upon follow-up, were found to heal normally or become nonunions. The samples were examined by immunohistochemistry to determine the expression of BMP-2, BMP-14, and the BMP inhibitors noggin and chordin. Expression was determined semiquantitatively based on the area of positive staining per area of cartilage and by determining the number of positively staining cells and the intensity of staining. There was a significant reduction in BMP-2 and BMP-14 expression in cartilaginous areas of nonhealing fractures compared to healing fractures. However, there was no difference in the expression of the BMP inhibitors between the two groups of fractures. This imbalance in the expression of BMPs and BMP inhibitors within cartilaginous areas of developing nonunions may account for their reduced bone forming ability. These data suggest strategies for preventing the development of nonunions by altering levels of BMPs and their inhibitors within fracture sites. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 752–757, 2009     

Expression and localization of ossification-related factors in fracture healing: Comparative study of experimental union and non-union models

The immunohistochemical expression and localization of some ossification-related factors, including a bone growth factor (bone morphogenetic protein, BMP), non-collagenous proteins (osteocalcin, BGP; bone sialoprotein, BSP), and type II collagen, were compared in union and non-union models of fracture healing in rats. In the union model, fracture healing was seen by day 25, whereas the non-union model did not show healing even on day 32. In both models, BMP, BGP, BSP, and type II collagen were expressed mainly in chondrocytes, with proliferating cell nuclear antigen (PCNA)-positive reaction in the soft callus. In the non-union model, the expression of BMP, BGP, BSP, type II collagen, and PCNA disappeared earlier than in the union model. In fracture healing, PCNA-positive cells expressed the ossification-related factors, these being essential for ossification.     

Effects of OP-1 and PTH in a new experimental model for the study of metaphyseal bone healing.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.     

骨质疏松性骨折愈合过程中的骨重建因子

一般性骨折愈合过程分为血肿机化演进期、原始骨痂形成期和骨痂改造塑形期;而骨质疏松性骨折愈合过程分为纤维骨痂期、软骨骨痂期和骨性骨痂期。骨质疏松性骨折愈合的组织学观察表明,纤维骨痂期主要是成纤维细胞合成Ⅲ型胶原;软骨骨痂期主要是成软骨细胞合成、分泌Ⅱ型胶原;骨性骨痂期主要是随着软骨内成骨的出现和发展,Ⅱ型胶原消失,逐渐由抗张力性能较强的Ⅰ型胶原所取代。一般来说,实验性骨质疏松性骨折的愈合方式与一般性骨折愈合方式相似,软骨内成骨和膜内成骨共同参与了骨质疏松性骨折的修复过程,但以软骨内成骨为主。     

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic “carrier” cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm‐long femur defect in a rat model occurs in under 3 weeks, through secretion of 100‐fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2–3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1597–1604, 2013     

锁定钢板联合自体松质骨、BMP治疗小儿股骨干骨折术后不愈合的疗效:生物相容性研究

目的探讨锁定钢板联合自体松质骨,骨形态发生蛋白(BMP)治疗小儿股骨干骨折不愈合的临床疗效。方法 2012年1月-2016年6月我院共收治28例小儿股骨干骨折术后延迟愈合患者,其中16例采用锁定钢板联合同种异体骨植骨和BMP治疗,12例采用弹性髓内钉固定。观察两组术中出血量、手术时间、住院时间、住院费用和术后下地锻炼时间,术后随访1年,定期复查X线片,观察骨折愈合情况和内固定稳定性,统计并发症发生情况。结果术后所有病例均达到骨性愈合,X线发现内固定无松动。观察组平均愈合时间为3.89±0.28个月,对照组为3.82±0.25个月,两组差异无统计学意义(P0.05)。在随访期间均未出现骨折不愈或延迟愈合。两组均未出现感染、股骨头坏死、髋内翻畸形、低血压等并发症。两组术中出血量、住院时间、住院费用和术后下地锻炼时间无明显差异(P0.05);手术时间对照组长于观察组,其差异有统计学意义(P0.05)。结论锁定钢板联合自体松质骨,骨形态发生蛋白治疗小儿股骨干骨折不愈合疗效可靠,并发症少。     

Low‐magnitude high‐frequency vibration enhances gene expression related to callus formation,mineralization and remodeling during osteoporotic fracture healing in rats

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col‐2), osteogenesis (Col‐1) and remodeling (receptor activator for nuclear factor‐ κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post‐treatment. LMHFV enhanced osteoporotic fracture healing by up‐regulating the expression of chondrogenesis‐, osteogenesis‐ and remodeling‐related genes (Col‐2 at week 4 (p = 0.008), Col‐1 at week 2 and 8 (p < 0.001and p = 0.008) and RANKL/OPG at week 8 (p = 0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col‐2 and Col‐1 at week 2 (p < 0.001 for all), larger callus width at week 2 (p = 0.001), callus area at week 1 and 5(p < 0.05 for all) and greater relative area of osseous tissue (p = 0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1572–1579, 2014.     

Osteogenic gene regulation and relative acceleration of healing by adenoviral‐mediated transfer of human BMP‐2 or ‐6 in equine osteotomy and ostectomy models

This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)‐2, Ad‐BMP‐6, or beta‐galactosidase protein vector control (Ad‐LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad‐BMP‐2 or Ad‐BMP‐6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad‐BMP‐2 than Ad‐BMP‐6, and both Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated osteotomies were greater than Ad‐LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta‐1, cathepsin H, and gelsolin‐like capping protein were greater in Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated callus compared to Ad‐LacZ‐treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad‐BMP‐2 over Ad‐BMP‐6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:764–771, 2008     

BMP‐6 and BMPR‐1a are up‐regulated in the growth plate of the fractured tibia

Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long‐term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture‐animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague–Dawley rats (male, 4 weeks old), were subjected to a unilateral mid‐diaphyseal tibial fracture. Kinetic expression of physeal BMP‐2, ‐4, ‐6, ‐7, and BMP receptor‐1a (BMPR‐1a) was analyzed in a monthly period by quantitative real time‐polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post‐fracture, no changes in physeal BMPs gene‐expression were detected. Twenty‐nine days post‐fracture, when the fracture was consolidated, physeal expression of BMP‐6 and BMPR‐1a was significantly upregulated in the growth plate of the fractured and contra‐lateral intact bone compared to control (p < 0.005). This study demonstrates a late role of BMP‐6 and BMPR‐1a in fracture‐induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory “cross‐talk” mechanism between the lower limbs whose function could be to limit leg‐length‐discrepancies following the breakage of growing bones. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 357–363, 2013     

Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing

BACKGROUND: Tissue ischemia and aging are independent features associated with the healing impairment of cutaneous wounds. However, the pathophysiology of these processes as they relate to impaired-healing wounds is poorly understood. MATERIALS AND METHODS: A single full-thickness biopsy wound was made on both ears of young (3-6 month) and aged (>24 month) Fisher rats. One ear was rendered ischemic by transection of the vasculature at the ear base, while the other ear served as an internal nonischemic control. Wounds were harvested from 3 to 7 days and were evaluated histologically for either granulation tissue formation and epithelialization. Total RNA from wounds harvested at postoperative day 7 was probed using a nylon-based cDNA array to assess global genetic expression alterations. RESULTS: Healing in the rat ear model is impaired by both ischemia and advanced age as measured by granulation tissue formation and wound epithelialization. Granulation tissue formation was affected to a greater degree by ischemia than age (-58% versus -21%, respectively) while epithelialization displayed an opposite response (-17% versus -53%, respectively). Global analysis of gene expression suggests that ischemia engenders a marked increase in genes displaying altered expression in aged animals compared to young animals. Importantly, all possible alterations in gene expression are found in samples from aged ischemic wounds, indicating that gene regulation is not simply depressed by advanced age. CONCLUSIONS: Wound epithelialization appears to be affected to a greater degree by advanced age than by ischemia. The results demonstrate the distinctive phenotype presented by the clinically relevant combination of age and ischemia in an in vivo model of cutaneous wound healing.     

Rotator cuff healing using demineralized cancellous bone matrix sponge interposition compared to standard repair in a preclinical canine model

This Level II study assessed clinically relevant outcomes for repair of large, retracted infraspinatus tendons (ISTs) using a demineralized bone matrix (DBM) sponge (FlexiGraft) hydrated in platelet‐rich plasma (PRP) versus direct repair in a validated canine model. Adult research dogs (n = 10) were used. The IST was transected in each shoulder (n = 20) and randomized to direct repair or repair with DBM‐PRP interposition at 4 weeks posttransection. At 12 weeks postrepair, dogs were sacrificed, and the repair evaluated by magnetic resonance imaging (MRI), histology, and biomechanical testing. MRI and histology scores were significantly (p < 0.05) better in the DBM‐PRP shoulders. Biomechanical testing revealed significantly improved strength of the DBM‐PRP repairs at 5 and 10 mm of displacement, as well as for ultimate failure load. In this canine model of retracted IST repair, DBM‐PRP sponge hydrated in PRP was considered safe and effective. In addition, use of DBM‐PRP was associated with improved MRI and histologic appearance, and improved strength compared to direct repair. Clinical significance: Based on reported failure rates for repair of large, retracted rotator cuff tears, improving tendon‐to‐bone healing is critical. Use of DBM combined with PRP shows potential for addressing this critical clinical need. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:906–912, 2018.

     

Microtomographic analysis of healing of femtosecond laser bone calvarial wounds compared to mechanical instruments in mice with and without application of BMP-7

BACKGROUND AND OBJECTIVE: This study investigated the healing of femtosecond laser created wounds in an animal model. STUDY DESIGN: We have assessed the healing of critical size wounds in mice calvaria using three different wounding techniques: carbide bur, diamond end-cutting bur, and ultrafast femtosecond laser, and in the presence or absence of bone morphogenetic protein-7 (BMP). Wound closure was examined using microcomputerized tomography at 3, 6, 9, and 12 weeks. RESULTS: Results have shown partial closure at up to 12 weeks with all techniques that did not involve the use of BMP, with the least closure noted in the laser groups as suggested by two-dimensional radiographic analysis. Bone volume measurements appeared slightly lower for the laser than for the mechanical groups, however statistically significant differences were seen only at week 6. No significant differences in closure were noted for the different methods in the BMP treated groups. CONCLUSIONS: Femtosecond laser cutting demonstrated an unsurpassed precision when compared to mechanical instruments. The addition of BMP led to very rapid healing with complete closure seen as early as 3 weeks and overcomes any potential healing delays that may arise from laser tissue cutting.     

Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.

This study evaluated healing of rabbit bilateral ulnar osteotomies 6 and 8 weeks after surgery in response to percutaneous injection of transgenic adenoviral (Ad) bone morphogenetic protein-6 (BMP-6) vector or green fluorescent protein vector control (Ad-GFP) administered 7 days after surgery compared to untreated osteotomy controls. The amount, composition and biomechanical properties of the healing bone repair tissue were compared among groups and to historical data for intact rabbit ulnae obtained from similar studies at the same institution. Quantitative computed tomography was used to determine area, density and mineral content of the mineralized callus in the harvested ulnae. Maximum torque, torsional stiffness, and energy absorbed to failure were determined at 1.5 degrees /s. Calcified sections of excised ulnae (5 microm) were stained with Goldner's Trichrome and Von Kossa, and evaluated for callus composition, maturity, cortical continuity, and osteotomy bridging. Radiographic assessment of bone formation indicated greater mineralized callus in the ulnae injected with Ad-hBMP-6 as early as 1 week after treatment (2 weeks after surgery) compared to untreated osteotomy ulnae (p < 0.006) and Ad-GFP treated osteotomy ulnae (p < 0.002). Quantitative computed tomography confirmed greater bone area and bone mineral content at the osteotomy at 6 weeks in Ad-BMP-6 treated osteotomy as compared to untreated osteotomy ulnae (p < 0.001) and Ad-GFP treated osteotomy ulnae (p < 0.01). Ad-BMP-6 treated osteotomy ulnae were stronger (p < 0.001 and 0.003) and stiffer (p < 0.004 and 0.003) in torsion at 6 weeks than untreated osteotomy ulnae or Ad-GFP treated osteotomy ulnae, respectively. Maximum torque, torsional stiffness, and energy absorbed to failure were greater in Ad-BMP-6 treated osteotomy ulnae compared to their respective untreated contralateral osteotomy ulnae at 8 weeks [p < 0.03]. Maximum torque and torsional stiffness in the Ad-BMP-6 treated osteotomy ulnae were not different to intact ulnae values at 6 and 8 weeks. These experiments confirm that BMP-6 can be potently osteoinductive in vivo resulting in acceleration of bone repair.     

Gene and protein expressions of bone marrow mesenchymal stem cells in a bone tunnel for tendon-bone healing

Purpose

Bone marrow mesenchymal stem cells (BMSCs) injected around tendon grafts can enhance tendon-bone healing and promote fibrocartilage formation. To understand gene and protein expressions of cells during tendon-bone healing, auto-BMSCs were implanted into a bone tunnel in anterior cruciate ligament reconstruction in a rabbit model.

Methods

BMSCs were harvested from New Zealand white rabbits. By an anterior cruciate ligament reconstruction model, 1 × 10⁶ BMSCs in 0.35 mL of fibrin glue was injected into bone tunnel as Fibrin-BMSC group. Only fibrin glue (Fibrin group) was injected as control. Three chondrogenesis genes and proteins, including Sox 9, collagen Type II (COII), aggrecan, and three osteogenesis genes and proteins, including Runx2, collagen type I (COI), and osteocalcin, between Fibrin-BMSC and Fibrin group were compared by real-time polymerase chain reaction assay and immunohistochemical assay postoperation.

Results

In real-time polymerase chain reaction assay, Sox9, COII, aggrecan, COI, and osteocalcin expressions upregulated and Runx2 downregulated were determined in Fibrin-BMSC group at 1 week. COII, aggrecan upregulated, and Runx2 and osteocalcin downregulated were determined at 4 weeks. In immunohistochemical assay, only Sox9, COII, and aggrecan proteins in only Fibrin-BMSC group were observed at 4 weeks. The protein expression as same as gene expression was obtained in a bone tunnel.

Conclusion

Auto-BMSCs promoted COII and aggrecan expression and reduced Runx2 and osteocalcin expression in a bone tunnel. It demonstrated that these cells could enhance fibrocartilage formation because of higher chondrogenesis expression during tendon-bone healing.     

In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models.

Regional gene therapy techniques are promising methods to enhance bone formation in large bone defects that would be difficult to treat with allograft or autograft bone stock. In this study, we compared in vivo temporal expression patterns of adenoviral- and lentiviral-mediated gene therapy in two bone formation models. Primary rat bone marrow cells (RBMC) were transduced with lentiviral or adenoviral vectors containing luciferase (Luc) or BMP-2 cDNA, or cotransduced with vectors containing Luc and bone morphogenetic protein 2 (BMP-2). In vitro protein production was determined with luciferase assay or ELISA (for BMP-2 production) weekly for 12 weeks. Two bone formation models were used -- a hind limb muscle pouch or radial defect -- in SCID mice. A cooled charged-coupled device (CCD) camera was used to image in vivo luciferase expression weekly for 12 weeks. In vitro, adenoviral expression of BMP-2 and luciferase was detected by ELISA or luciferase assay, respectively, for 4 weeks. Lentiviral expression of BMP-2 and luciferase was sustained in culture for 3 months. Using the CCD camera, we found that adenoviral vectors expressed luciferase expression for up to 21 days, but lentiviral vectors expressed target gene expression for 3 months in vivo in both bone formation models. There was no detectable difference in the amount of bone formed between the adenoviral and lentiviral groups. Lentiviral-mediated delivery of BMP-2 can induce long term in vitro and in vivo gene expression, which may be beneficial when developing tissue engineering strategies to heal large bone defects or defects with a compromised biologic environment.     

Development of an atrophic nonunion model and comparison to a closed healing fracture in rat femur.

Although most fractures heal, some fail to heal and become nonunions. Many animal models have been developed to study problems of fracture healing. The majority of nonunion models have involved segmental bone defects, but this may not adequately represent the biologic condition in which nonunions clinically develop. The objective of the present study is to develop a nonunion model that better simulates the clinical situation in which there is soft tissue damage including periosteal disruption and to compare this model to a standard closed fracture model utilizing identical fracture stabilization, providing a similar mechanical environment. A total of 96 three month old Long Evans rats were utilized. A 1.25 mm diameter K-wire was inserted into the femur in a retrograde fashion, and a mid-diaphyseal closed transverse fracture was created using a standard three-point bending device. To create a nonunion, 48 of the rats received additional surgery to the fractured femur. The fracture site was exposed and 2 mm of the periosteum was cauterized on each side of the fracture. Fracture healing was evaluated with serial radiographs every two weeks. Animals were maintained for intervals of two, four, six or eight weeks after surgery. Specimens from each time interval were subjected to biomechanical and histological evaluation. None of the cauterized fractures healed throughout the eight weeks experimental duration. The radiographical appearance of nonunion models was atrophic. This investigation showed pronounced differences between the experimental nonunions and standard closed fractures both histologically and biomechanically. In conclusion, we have developed a reproducible atrophic nonunion model in the rat femur that simulates the clinical condition in which there is periosteal disruption but no bone defect.     

The experimental agent pirfenidone reduces pro-fibrotic gene expression in a model of tacrolimus-induced nephrotoxicity

BACKGROUND: Tacrolimus nephrotoxicity is thought to contribute to renal allograft dysfunction and subsequent failure, a process that is underpinned by alterations in mRNA expression of genes involved in matrix metabolism. The new anti-fibrotic pirfenidone was tested for its potential to reverse markers of renal dysfunction. MATERIALS AND METHODS: Rats were salt-depleted before tacrolimus and pirfenidone treatment. Serum creatinine, urinary protein/creatinine ratio, extracellular matrix deposition (ECM), and mRNA expression of genes involved in matrix turnover were assessed. RESULTS: Tacrolimus reduced TGF-beta mRNA expression below control levels and treatment with pirfenidone at all doses did not alter this effect. Likewise, TIMP-1 mRNA expression was depressed by the addition of tacrolimus and pirfenidone caused a further decrease in expression. Collagen III, MMP-2, and MMP-9 expression was unchanged by tacrolimus, but pirfenidone reduced collagen III below control levels. ECM was slight (1-4%) and not significantly different between groups. CONCLUSIONS: These findings suggest that pirfenidone can attenuate the limited fibrotic potential of tacrolimus.