The role of muscle loading on bone (Re)modeling at the developing enthesis.

Muscle forces are necessary for the development and maintenance of a mineralized skeleton. Removal of loads leads to malformed bones and impaired musculoskeletal function due to changes in bone (re)modeling. In the current study, the development of a mineralized junction at the interface between muscle and bone was examined under normal and impaired loading conditions. Unilateral mouse rotator cuff muscles were paralyzed using botulinum toxin A at birth. Control groups consisted of contralateral shoulders injected with saline and a separate group of normal mice. It was hypothesized that muscle unloading would suppress bone formation and enhance bone resorption at the enthesis, and that the unloading-induced bony defects could be rescued by suppressing osteoclast activity. In order to modulate osteoclast activity, mice were injected with the bisphosphonate alendronate. Bone formation was measured at the tendon enthesis using alizarin and calcein fluorescent labeling of bone surfaces followed by quantitative histomorphometry of histologic sections. Bone volume and architecture was measured using micro computed tomography. Osteoclast surface was determined via quantitative histomorphometry of tartrate resistant acid phosphatase stained histologic sections. Muscle unloading resulted in delayed initiation of endochondral ossification at the enthesis, but did not impair bone formation rate. Unloading led to severe defects in bone volume and trabecular bone architecture. These defects were partially rescued by suppression of osteoclast activity through alendronate treatment, and the effect of alendronate was dose dependent. Similarly, bone formation rate was increased with increasing alendronate dose across loading groups. The bony defects caused by unloading were therefore likely due to maintained high osteoclast activity, which normally decreases from neonatal through mature timepoints. These results have important implications for the treatment of muscle unloading conditions such as neonatal brachial plexus palsy, which results in shoulder paralysis at birth and subsequent defects in the rotator cuff enthesis and humeral head.

CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis.

Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.

A role for PACE4 in osteoarthritis pain: evidence from human genetic association and null mutant phenotype.

OBJECTIVES: The aim of this study was to assess if genetic variation in the PACE4 (paired amino acid converting enzyme 4) gene Pcsk6 influences the risk for symptomatic knee osteoarthritis (OA). METHODS: Ten PCSK6 single nucleotide polymorphisms were tested for association in a discovery cohort of radiographic knee OA (n=156 asymptomatic and 600 symptomatic cases). Meta-analysis of the minor allele at rs900414 was performed in three additional independent cohorts (total n=674 asymptomatic and 2068 symptomatic). Pcsk6 knockout mice and wild-type C57BL/6 mice were compared in a battery of algesiometric assays, including hypersensitivity in response to intraplantar substance P, pain behaviours in response to intrathecal substance P and pain behaviour in the abdominal constriction test. RESULTS: In the discovery cohort of radiographic knee OA, an intronic single nucleotide polymorphism at rs900414 was significantly associated with symptomatic OA. Replication in three additional cohorts confirmed that the minor allele at rs900414 was consistently increased among asymptomatic compared to symptomatic radiographic knee OA cases in all four cohorts. A fixed-effects meta-analysis yielded an OR=1.35 (95% CI 1.17 to 1.56; p=4.3×10(-5) and no significant between-study heterogeneity). Studies in mice revealed that Pcsk6 knockout mice were significantly protected against pain in a battery of algesiometric assays. CONCLUSIONS: These results suggest that a variant in PCSK6 is strongly associated with protection against pain in knee OA, offering some insight as to why, in the presence of the same structural damage, some individuals develop chronic pain and others are protected. Studies in Pcsk6 null mutant mice further implicate PACE4 in pain.

Intraarticular injection of hyaluronan prevents cartilage erosion, periarticular fibrosis and mechanical allodynia and normalizes stance time in murine knee osteoarthritis.

INTRODUCTION: Intraarticular hyaluronan (HA) is used clinically for symptomatic relief in patients with knee osteoarthritis (OA); however, the mechanism of action is unclear. In this study, we examined the effects of a single injection of HA on joint tissue pathology, mechanical allodynia and gait changes (measured by stride times) in a murine model of OA. METHODS: OA was induced in the right knee joint (stifle) of 12-week-old male C57BL/6 mice by transforming growth factor ß1 (TGFß1) injection and treadmill running for 14 days. Gait parameters were quantified by using TreadScan, mechanical allodynia was evaluated with von Frey filaments, and joint pathology was evaluated by scoring of macroscopic images for both cartilage erosion and periarticular fibrosis. HA or saline control was injected 1 day after TGFß1 injection but before the start of treadmill running. RESULTS: OA development in this model was accompanied by significant (P < 0.01) enhancement of the stance and propulsion times of affected legs. HA injection (but not saline injection) blocked all gait changes and also protected joints from femoral cartilage erosion as well as tibial and femoral tissue fibrosis. Both HA injection and saline injection attenuated acute allodynia, but the HA effect was more pronounced and prolonged than the saline injection. CONCLUSIONS: We conclude that videographic gait analysis is an objective, sensitive and reproducible means of monitoring joint pathology in experimental murine OA, since stance time appears to correlate directly with OA severity. A single injection of HA prevents acute and prolonged gait changes and ameliorates the cartilage erosion and periarticular fibrosis normally seen in this model. We speculate that the capacity of HA to prevent cartilage erosion results from its normalization of joint biomechanics and its inhibitory effects on periarticular cells, which are involved in tissue hyperplasia and fibrosis. This effect of exogenous HA appears to mimic the protective effects of ablation of Adamts5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) on experimental murine OA, and we speculate that a common mechanism is involved.

Effects of botulinum toxin-induced paralysis on postnatal development of the supraspinatus muscle.

The mechanical environment plays an important role in musculoskeletal tissue development. The present study characterized changes in supraspinatus muscle due to removal of mechanical cues during postnatal development. An intramuscular injection of botulinum toxin type A (BTX) was used to induce and maintain paralysis in the left shoulders of mice since birth while the right shoulders received saline and served as contralateral controls. A separate group of animals was allowed to develop normally without any injections. Muscles were examined postnatally at various time points. The maximum isometric tetanic force generated by the muscle was significantly reduced in the BTX group compared to saline and normal groups. The paralyzed muscles were smaller and showed significant muscle atrophy and fat accumulation on histologic evaluation. Myogenic genes myogenin, myoD1, myf5, myf6, and fast type II myosin heavy chain (MHC) isoform were significantly upregulated while slow type I MHC isoform was significantly downregulated in the BTX group. Adipogenic genes C/EBPα, PPARγ2, leptin, and lipoprotein lipase were significantly upregulated in the BTX group. Results indicate that reduced muscle loading secondary to BTX-induced paralysis leads to fat accumulation and muscle degeneration in the developing muscle. Understanding the molecular and compositional changes in developing supraspinatus muscles may be useful for identifying and addressing the pathological changes that occur in shoulder injuries such as neonatal brachial plexus palsy.

Fibrocartilage tissue engineering: the role of the stress environment on cell morphology and matrix expression.

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development.

The role of transforming growth factor beta isoforms in tendon-to-bone healing.

The purpose of this study was to examine the role of two of the three transforming growth factor beta (TGF-ß) isoforms at the healing tendon-to-bone insertion. The supraspinatus tendons of 64 rats were transected at their bony insertions and repaired to the humeral head. One shoulder of each rat received an osmotic pump for sustained delivery of the following factors at the repair site: (1) TGF-ß1 and neutralizing antibodies to TGF-ß2 and 3 (TGF-ß1 group), (2) TGF-ß3 and neutralizing antibodies to TGF-ß1 and 2 (TGF-ß3 group), (3) neutralizing antibodies to TGF-ß1, 2, and 3 (anti-TGF-ß group), and (4) saline (saline group). The contralateral shoulders received saline to serve as paired controls. The repairs were evaluated at multiple time points postmortem using histology-based assays and biomechanical testing. Treated shoulders in the TGF-ß1 group showed increased type III collagen production compared to the paired control shoulders, indicative of a scar-mediated response. There was a trend toward reduced mechanical properties in the TGF-ß1 group, but these changes did not reach statistical significance. The anti-TGF-ß group showed no difference in tissue volume, but significantly inferior mechanical properties, compared to the paired control shoulders. The TGF-ß3 group did not show any differences compared to the paired control shoulders. Although TGF-ß isoforms play important roles in tendon-to-bone development and healing, application of exogenous TGF-ß isoforms and neutralizing antibodies to the subacromial space using osmotic pumps did not improve supraspinatus tendon-to-bone healing.

The effects of exogenous basic fibroblast growth factor on intrasynovial flexor tendon healing in a canine model.

BACKGROUND: Studies have demonstrated that flexor tendon repair strength fails to increase in the first three weeks following suturing of the tendon, a finding that correlates closely with the timing of many clinical failures. The application of growth factors holds promise for improving the tendon-repair response and obviating failure in the initial three weeks. METHODS: The effects of basic fibroblast growth factor on flexor tendon healing were evaluated with use of a canine model. Operative repair followed by the sustained delivery of basic fibroblast growth factor, at two different doses, was compared with operative repair alone. Histological, biochemical, and biomechanical methods were used to evaluate the tendons twenty-one days after repair. RESULTS: Vascularity, cellularity, and adhesion formation were increased in the tendons that received basic fibroblast growth factor as compared with the tendons that received operative repair alone. DNA concentration was increased in the tendons that received 1000 ng of basic fibroblast growth factor (mean and standard deviation, 5.7 ± 0.7 µg/mg) as compared with the tendons that received 500 ng of basic fibroblast growth factor (3.8 ± 0.7 µg/mg) and the matched control tendons that received operative repair alone (4.5 ± 0.9 µg/mg). Tendons that were treated with basic fibroblast growth factor had a lower ratio of type-I collagen to type-III collagen, indicating increased scar formation compared with that seen in tendons that received operative repair alone (3.0 ± 1.6 in the group that received 500-ng basic fibroblast growth factor compared with 4.3 ± 1.0 in the paired control group that received operative repair alone, and 3.4 ± 0.6 in the group that received 1000-ng basic fibroblast growth factor compared with 4.5 ± 1.9 in the paired control group that received operative repair alone). Consistent with the increases in adhesion formation that were seen in tendons treated with basic fibroblast growth factor, the range of motion was reduced in the group that received the higher dose of basic fibroblast growth factor than it was in the paired control group that received operative repair alone (16.6° ± 9.4° in the group that received 500 ng basic fibroblast growth factor, 13.4° ± 6.1° in the paired control group that received operative repair alone, and 29.2° ± 5.8° in the normal group [i.e., the group of corresponding, uninjured tendons from the contralateral forelimb]; and 15.0° ± 3.8° in the group that received 1000 ng basic fibroblast growth factor, 19.3° ± 5.5° in the paired control group that received operative repair alone, and 29.0° ± 8.8° in the normal group). There were no significant differences in tendon excursion or tensile mechanical properties between the groups that were treated with basic fibroblast growth factor and the groups that received operative repair alone. CONCLUSIONS: Although basic fibroblast growth factor accelerated the cell-proliferation phase of tendon healing, it also promoted neovascularization and inflammation in the earliest stages following the suturing of the tendon. Despite a substantial biologic response, the administration of basic fibroblast growth factor failed to produce improvements in either the mechanical or functional properties of the repair. Rather, increased cellular activity resulted in peritendinous scar formation and diminished range of motion.

Technical and biological modifications for enhanced flexor tendon repair.

Clinical outcomes after intrasynovial flexor tendon repair have been substantially improved over the past 2 decades through advances in tendon suture techniques and postoperative rehabilitation methods. Nevertheless, complications such as repair site elongation (i.e., gap formation) and rupture continue to occur frequently. Experimental studies have shown that repair site strength fails to increase in the first 3 weeks after tendon suture. After 3 weeks, the strength and rigidity of the repair site improve significantly, a process that continues for several months. Formation of a repair site gap during the early rehabilitation period has been shown to considerably delay the accrual of repair site strength over time. Thus, it is of prime importance that the method of tendon suture achieves and maintains a stiff and strong repair site during the early healing interval by maintaining close approximation of the tendon stumps and by stimulating, where possible, the intrinsic repair response. In this review, we describe recent efforts to enhance the integrity of the immature repair site. We focus on 2 major areas of advancement: surgical technique modifications and manipulation of the biologic and biochemical environment.

Musculoskeletal deformities secondary to neurotomy of the superior trunk of the brachial plexus in neonatal mice.

The developmental course of musculoskeletal deformities in neonatal brachial plexus palsy (NBPP) has not been studied extensively. The goals of this study were to: (1) evaluate a new animal model of NBPP, (2) characterize the development of musculoskeletal abnormalities in paralyzed shoulders, and (3) investigate the expression of myogenic and adipogenic genes in paralyzed rotator cuff muscles. Neonatal mice were divided into neurotomy and sham groups. The neurotomy group underwent surgical transection of the superior trunk of the brachial plexus within 24 h of birth. The sham group underwent the same surgical exposure, but the brachial plexus was left intact. Musculoskeletal deformities were evaluated with radiological and histological assays at 2, 4, 8, 12, and 30 weeks after birth. The supraspinatus muscles of a separate group of mice were used to examine expression of myogenic and adipogenic genes at 8 weeks. The neurotomized forelimbs developed deformities similar to those seen in human NBPP. The deformities progressed with age. The denervated supraspinatus muscles showed intramuscular fat accumulation and upregulation of both myogenic and adipogenic genes compared to the normal. The current study presents a useful animal model for future research examining musculoskeletal changes secondary to neonatal nerve injury.

bFGF and PDGF-BB for tendon repair: controlled release and biologic activity by tendon fibroblasts in vitro.

Flexor tendon injuries are often encountered clinically and typically require surgical repair. Return of function after repair is limited due to adhesion formation, which leads to reduced tendon gliding, and due to a lack of repair site strength, which leads to repair site gap formation or rupture. The application of the growth factors basic fibroblastic growth factor (bFGF) and platelet derived growth factor BB (PDGF-BB) has been shown to have the potential to enhance tendon healing. The objectives of this study were to examine: (1) the conditions over which delivery of bFGF can be controlled from a heparin-binding delivery system (HBDS) and (2) the effect of bFGF and PDGF-BB released from this system on tendon fibroblast proliferation and matrix gene expression in vitro over a 10-day interval. Delivery of bFGF was controlled using a HBDS. Fibrin matrices containing the HBDS retained bFGF better than did matrices lacking the delivery system over the 10-day period studied. Delivery of bFGF and PDGF-BB using the HBDS stimulated tendon fibroblast proliferation and promoted changes in the expression of matrix genes related to tendon gliding, strength, and remodeling. Both growth factors may be effective in enhancing tendon healing in vivo.

Use of a magnesium-based bone adhesive for flexor tendon-to-bone healing.

PURPOSE: Our previous studies in a canine animal model demonstrated that the flexor tendon-to-bone insertion site has a poor capacity to heal. Magnesium-based adhesives have the potential to improve tendon-to-bone healing. Therefore, we hypothesized that magnesium-based bone adhesive (MBA) will improve the tendon-to-bone biomechanical properties initially and in the early period after repair. METHODS: Flexor digitorum profundus tendons were injured and repaired into bone tunnels in the distal phalanges of dogs. The bone tunnels were either filled with MBA before completing the repair or left empty (control [CTL]). Histologic appearance, tensile properties, range of motion, and bone density were examined at time zero and 21 days after the repair. RESULTS: There was no histologic evidence of acute inflammation. There appeared to be more mast cells in the MBA group than in the CTL group. Chronic inflammatory infiltrate and fibrosis was slightly higher in the MBA group compared with the CTL group. Tensile properties at time zero were significantly higher in the MBA group compared with the CTL group. However, tensile properties were significantly lower in the MBA group compared with the CTL group at 21 days. Range of motion and bone density were significantly lower in the MBA and CTL groups compared with normal (ie, uninjured) at 21 days; no differences were seen when comparing MBA with CTL. CONCLUSIONS: We found that the initial biomechanical properties of flexor tendon-to-bone repairs can be improved with MBA. However, MBA use in vivo led to a decrease in the biomechanical properties of the repair. There was no effect of MBA on bone density or range of motion in the early period after repair. Our histologic analysis suggests that the poor healing in the MBA group may have been due to an allergic response or to increased chronic inflammation resulting from the foreign material.

Complete removal of load is detrimental to rotator cuff healing.

HYPOTHESIS: This study evaluated the effect of the mechanical environment on the healing rotator cuff by paralyzing the supraspinatus muscle in the operative shoulder of a rat model of rotator cuff injury and repair. METHODS: Unilateral shoulders of rats underwent a supraspinatus injury and repair. Botulinum toxin A was used to paralyze the muscle after repair. Postoperatively, 1 group was immobilized and 1 group was allowed free range of motion. Saline-injected, casted rats were used as the control group. Repairs were evaluated histologically, geometrically, and biomechanically. RESULTS: Specimens from the saline-injected rats had greater scar volume and cross-sectional area of the repair compared with the paralyzed groups. Structural properties were increased in the saline group compared with the paralyzed groups. Free range of motion (ie, uncasted group) resulted in modest improvements in biomechanical properties but did not obviate the effect of paralysis. CONCLUSIONS: Complete removal of load was detrimental to rotator cuff healing, especially when combined with immobilization.

Recovery potential after postnatal shoulder paralysis. An animal model of neonatal brachial plexus palsy.

BACKGROUND: Injury to the brachial plexus during birth results in paralysis of the upper extremity in as many as one in 250 births and can lead to substantial functional deficits in the shoulder. The goal of this study was to characterize the development of bone and joint deformities in paralyzed neonatal shoulders and to assess the improvement of these deformities after muscle function recovery with use of an animal model. METHODS: Intramuscular injections of botulinum toxin were used to paralyze the supraspinatus, infraspinatus, and posterior deltoid of the left shoulders of mice at birth. Seventy mice were divided into three groups: Botox, recovery, and normal. The twenty-five mice in the Botox group received botulinum toxin injections until they were killed. The twenty mice in the recovery group received botulinum toxin injections for different durations and then were allowed injection-free recovery periods until they were killed. The twenty-five mice in the normal group received saline solution injections until they were killed. Radiographs were used to measure shoulder and elbow contractures. Microcomputed tomography was used to examine anatomical parameters of the supraspinatus muscle, humerus, and scapula. RESULTS: The Botox group showed bone and joint deformities including delayed mineralization and flattening of the humeral head, hypoplasia, and introversion (i.e., anteversion) of the humerus, contractures of the shoulder and elbow, hypoplasia of shoulder muscles, hypoplasia of the scapula, and hypoplasia and retroversion of the glenoid. In the recovery group, a significant trend toward normal properties was observed with longer recovery periods (p<0.05). However, only soft-tissue contractures of the shoulder and elbow were resolved completely with the longest recovery period. CONCLUSIONS: This mouse model successfully simulates human neonatal brachial plexus palsy, reproducing most of the bone and joint deformities found in the human condition. The deformities started to develop early in the postnatal period in the paralyzed shoulders and progressed with longer durations of paralysis. Early restoration of muscle function completely resolved the soft-tissue contractures of the shoulder and elbow. However, osseous deformities of the humerus and scapula were never resolved completely. These findings demonstrate the time-dependence of reversibility of musculoskeletal deformities in developing shoulders with neurological deficits.

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.

Controlled-release kinetics and biologic activity of platelet-derived growth factor-BB for use in flexor tendon repair.

PURPOSE: Surgically repaired intrasynovial tendons are at greatest risk of failure in the first 3 weeks after surgery. Attempts to improve the strength of repair by modifying rehabilitation parameters have not always been successful. Manipulation of the biological environment of the sutured tendon holds great promise for accelerating the repair process. The goals of this study were to examine (1) the range of conditions (eg, dosage, delivery system formulation, presence of cells) over which delivery of platelet-derived growth factor-BB (PDGF-BB) can be sustained from fibrin matrices using a heparin-binding delivery system (HBDS) and (2) the biological activity of the PDGF-BB released from this system on canine tendon fibroblasts in vitro. METHODS: We examined in vitro release kinetics from cellular and acellular fibrin matrices using enzyme-linked immunosorbent assays. We examined the biologic activity of the PDGF-BB in vitro by measuring cell proliferation (ie, total DNA) and collagen synthesis (ie, proline incorporation). RESULTS: The acellular release kinetics of PDGF-BB was modulated by varying the ratio of PDGF-BB to heparin (PDGF-binding sites) or the dose of PDGF-BB in the presence of the delivery system. In the presence of canine tendon fibroblasts, the delivery system prolonged the duration of PDGF-BB release from fibrin matrices, thus demonstrating that cells are able to liberate PDGF-BB retained by the HBDS. Sustained delivery of PDGF-BB promoted increased cell proliferation at doses of 0.125 microg/mL and 1.25 microg/mL compared to fibrin without delivery system. Collagen synthesis was enhanced by PDGF-BB at doses of 0.125 microg/mL and 1.25 microg/mL; however, there was an enhancement over fibrin without the delivery system only at the lower dose. CONCLUSIONS: These results demonstrate that the PDGF-BB released from fibrin matrices containing an HBDS is biologically active and can modulate both cell proliferation and extracellular matrix synthesis, both of which are key factors in the process of tendon repair.

The effect of muscle loading on flexor tendon-to-bone healing in a canine model.

Previous tendon and ligament studies have demonstrated a role for mechanical loading in tissue homeostasis and healing. In uninjured musculoskeletal tissues, increased loading leads to an increase in mechanical properties, whereas decreased loading leads to a decrease in mechanical properties. The role of loading on healing tissues is less clear. We studied tendon-to-bone healing in a canine flexor tendon-to-bone injury and repair model. To examine the effect of muscle loading on tendon-to-bone healing, repaired tendons were either cut proximally (unloaded group) to remove all load from the distal phalanx repair site or left intact proximally (loaded group). All paws were casted postoperatively and subjected to daily passive motion rehabilitation. Specimens were tested to determine functional properties, biomechanical properties, repair-site gapping, and bone mineral density. Loading across the repair site led to improved functional and biomechanical properties (e.g., stiffness for the loaded group was 8.2 +/- 3.9 versus 5.1 +/- 2.5 N/mm for the unloaded group). Loading did not affect bone mineral density or gapping. The formation of a gap between the healing tendon and bone correlated with failure properties. Using a clinically relevant model of flexor tendon injury and repair, we found that muscle loading was beneficial to healing. Complete removal of load by proximal transection resulted in tendon-to-bone repairs with less range of motion and lower biomechanical properties compared to repairs in which the muscle-tendon-bone unit was left intact.

PDGF-BB released in tendon repair using a novel delivery system promotes cell proliferation and collagen remodeling.

The purpose of this study was to promote fibroblast proliferation and collagen remodeling in flexor tendon repair through sustained delivery of platelet derived growth factor (PDGF-BB). The release kinetics of PDGF-BB from a novel fibrin matrix delivery system was initially evaluated in vitro. After the in vivo degradation rate of the fibrin matrix was determined using fluorescently tagged fibrin, PDGF-BB was delivered to the site of flexor tendon repair in vivo in a canine model. The effect of PDGF-BB on intrasynovial tendon healing was studied using histology-based assays (cell density, proliferation, and type I collagen expression) and by measuring total DNA levels and reducible collagen crosslink levels. The fibrin matrix delivery system provided sustained release of PDGF-BB in vitro at a rate modulated by the ratio of heparin to growth factor. In vivo, the fibrin matrix remained at the repair site for more than 10 days. Delivery of PDGF-BB led to a qualitative increase in cell density, cell proliferation, and type I collagen mRNA expression. PDGF-BB also led to statistically significant increases in total DNA (20% increase at 7 days, 18% increase at 14 days) and reducible collagen crosslinks (30% increase at 7 days). Sustained delivery of growth factors may be achieved using a novel fibrin-based delivery system. PDGF-BB delivery increased cell proliferation and matrix remodeling and thus may accelerate flexor tendon healing.

Decreased muscle loading delays maturation of the tendon enthesis during postnatal development.

Physical environment influences the development and maintenance of musculoskeletal tissues. The current study uses an animal model to explore the role of the physical environment on the postnatal development of the supraspinatus tendon enthesis. A supraspinatus intramuscular injection of botulinum toxin A was used to paralyze the left shoulders of mice at birth. The supraspinatus muscles of right shoulders were injected with saline to serve as contralateral controls. The supraspinatus enthesis was examined after 14, 21, 28, and 56 days of postnatal development. Histologic assays were used to examine fibrocartilage morphology and percentage osteoclast surface. Micro-computed tomography was used to examine muscle geometry and bone architecture. At 14 days there were no differences between groups in fibrocartilage formation, muscle geometry, bone architecture, or osteoclast surface. When comparing groups at 21, 28, and 56 days, muscle volume was decreased, fibrocartilage development was delayed, mineralized bone was decreased, and osteoclast surface was higher at each timepoint in the botulinum group compared to the contralateral saline control group. Our results indicate that the development of the tendon enthesis is sensitive to its mechanical environment. A reduction in muscle loading delayed the development of the tendon-to-bone insertion site by impeding the accumulation of mineralized bone. Physical factors did not play a significant role in enthesis maturation in the first 14 days postnatally, implying that biologic factors may drive early postnatal development.

The early effects of sustained platelet-derived growth factor administration on the functional and structural properties of repaired intrasynovial flexor tendons: an in vivo biomechanic study at 3 weeks in canines.

PURPOSE: A bioactive fibrin-based delivery system was used to provide sustained administration of platelet-derived growth factor (PDGF-BB) in a clinically relevant model of intrasynovial flexor tendon repair. We hypothesized that PDGF-BB administered in this manner would improve the sutured tendon's functional and structural properties 3 weeks after repair. METHODS: A delivery system consisting of 30 microL of fibrin matrix, peptide, heparin, and 100 ng of PDGF-BB was incorporated into the repair sites of randomly selected medial or lateral forepaw flexor digitorum profundus tendons of 8 adult mongrel dogs. The remaining forepaw flexor digitorum profundus tendons were repaired without the growth-factor and fibrin-based delivery system and served as controls. The surgically treated forelimbs were treated with controlled passive motion rehabilitation. The animals were killed at 3 weeks, at which time the tendons were tested for range of motion with a motion analysis system and for tensile properties with a materials testing machine. RESULTS: Proximal interphalangeal joint and distal interphalangeal joint rotation values were significantly higher for the PDGF-BB-treated tendons compared with the repair-alone tendons. Excursion values were also significantly higher in the PDGF-BB-treated tendons. There were no significant differences in tensile properties when comparing PDGF-BB-treated with repair-alone tendons. CONCLUSIONS: The functional properties of repaired intrasynovial flexor tendons were significantly improved with the sustained administration of PDGF-BB. The failure to achieve improvements in ultimate load, stiffness, and strain in the experimental group may have been due to suboptimal PDGF-BB dosage or suboptimal release kinetics.