Rosalina Das

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.

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 tendons 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.

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.

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.

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 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-β) 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-β1 and neutralizing antibodies to TGF‐β2 and 3 (TGF-β1 group), (2) TGF-β3 and neutralizing antibodies to TGF-β1 and 2 (TGF-β3 group), (3) neutralizing antibodies to TGF-β1, 2, and 3 (anti-TGF-β 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-β1 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-β1 group, but these changes did not reach statistical significance. The anti-TGF-β group showed no difference in tissue volume, but significantly inferior mechanical properties, compared to the paired control shoulders. The TGF-β3 group did not show any differences compared to the paired control shoulders. Although TGF-β isoforms play important roles in tendon-to-bone development and healing, application of exogenous TGF-β isoforms and neutralizing antibodies to the subacromial space using osmotic pumps did not improve supraspinatus tendon-to-bone healing.

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.

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. Published by Wiley Periodicals, Inc. J Orthop Res 28:1391–1398, 2010

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.