Steven P. Arnoczky

To determine if tendon cell ciliary length could be used as a biomarker of cytoskeletal tensional homeostasis, 20 mm long adult rat tail tendons were placed in double artery clamps set 18 mm apart to create a 10% laxity. The tendons were allowed to contract over a 7 day period under culture conditions. At 0, 1, 5, and 7 days the tendon cell cilia were stained and ciliary length measured using confocal imaging. There was a significant (p<0.001) increase in ciliary length at 1 day. At day 5 (when the tendon became visibly taut) there was a significant (p<0.001) decrease in ciliary length compared to day 1. By day 7 the tendon remained taut and ciliary length returned to day zero values (p=0.883). These results suggest that cilia length reflects the local mechanobiological environment of tendon cells and could be used as a potential in situ biomarker of altered cytoskeletal tensional homeostasis.

To determine if an absorbable collagen scaffold of high porosity would allow rapid tissue in-growth and permit the functional maturation and alignment of tendon-like tissue, scaffolds were sutured to the superficial surface of the infraspinatus tendons of adult sheep. Histology demonstrated complete ingrowth with fibrovascular tissue by 6 weeks and by 12 weeks the scaffold had induced the formation of a layer of dense, regularly-oriented collagenous tissue which significantly increased the thickness of the native tendon. This new tissue was well-integrated into the host tissues at both the bone interface and along the length of the tendon. At 26 weeks the scaffold was completely absorbed leaving a stable layer of mature tendon-like tissue over the surface of the host tendon which was still present at 52 weeks. The use of a reconstituted collagen scaffold consistently increased the thickness of a rotator cuff tendon by inducing the formation of a well-integrated and mature tendon-like tissue.

Collagen crimp morphology is thought to contribute to the material behavior of tendons and may reflect the local mechanobiological environment of tendon cells. Following loss of collagen tension in tendons, tenocytes initiate a contraction response that shortens tendon length which, in turn, may alter crimp patterns. We hypothesized that changes in the crimp pattern of tendons are the result of cell‐based contractions which are governed by relative tautness/laxity of the collagen matrix. To determine the relationship between crimp pattern and tensional homeostasis, rat tail tendon fascicles (RTTfs) were either allowed to freely contract or placed in clamps with 10% laxity for 7 days. The freely contracting RTTfs showed a significant decrease in percent crimp length on both day 5 (3.66%) and day 7 (7.70%). This decrease in crimp length significantly correlated with the decrease in freely contracting RTTf length. Clamped RTTfs demonstrated a significant decrease in percent crimp length on day 5 (1.7%), but no significant difference in percent crimp length on day 7 (0.57%). The results demonstrate that the tendon crimp pattern appears to be under cellular control and is a reflection of the local mechanobiological environment of the extracellular matrix. The ability of tenocytes to actively alter the crimp pattern of collagen fibers also suggests that tenocytes can influence the viscoelastic properties of tendon. Understanding the interactions between tenocytes and their extracellular matrix may lead to further insight into the role tendon cells play in maintaining tendon heath and homeostasis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:573–579, 2017.

Purpose: To histologically evaluate biopsy specimens from patients who previously underwent rotator cuff repair augmented with a highly porous collagen implant. Methods: Biopsies of collagen implant/host‐tissue constructs were obtained from 7 patients undergoing a second arthroscopic procedure at various time periods (5 weeks to 6 months) after arthroscopic rotator cuff repair augmented with a collagen implant overlay. The biopsy specimens were examined histologically for host‐tissue ingrowth, host‐tissue maturation, and host‐implant biocompatibility. Results: At the earliest time period (5 weeks), the biopsy revealed the presence of host cells (fibroblasts) within the interstices of the porous collagen implant. Cells were aligned along the linear orientation of the collagen implant structure, and there was evidence of early collagen formation. The 3‐month biopsies showed increased collagen formation, maturation, and organization over the surface of the implant and evidence of the collagen implant. At 6 months, the newly generated tissue had the histologic appearance of a tendon, suggesting functional loading of the new generated host tissue. There was no evidence of any remnants of the collagen implant in the 6‐month biopsy. There was no evidence of any inflammatory or foreign body reaction within any of the tissue samples. Conclusions: Biopsies of collagen implants retrieved from human rotator cuff repair subjects revealed cellular incorporation, tissue formation and maturation, implant resorption, and biocompatibility. Clinical Relevance: The histologic observations from these clinical biopsies support the biocompatibility of this implant and its ability to promote new connective tissue with the histological appearance of tendon over the surface of the native cuff tendon.

Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder of collagen resulting in fragile, hyper-extensible skin and ulcerative lesions. The predominance of skin lesions have been shown to occur on the dorsum of HERDA-affected horses. While this has been postulated to be due to increased exposure to sunlight of these areas, the precise pathological mechanism which causes this to occur is unclear. We hypothesized that an increase in collagenase activity, that has been associated with the exposure of dermal fibroblasts to sunlight, will significantly degrade the material properties of skin from HERDA-affected horses when compared to unaffected controls. Six unaffected and seven HERDA-affected horses, all euthanized for other reasons. Full-thickness skin samples from similar locations on each horse were collected and cut into uniform strips and their material properties (tensile modulus) determined by mechanical testing before (n = 12 samples/horse) or after (n = 12 samples/horse) incubation in bacterial collagenase at 37°C for 6 h. The change in modulus following treatment was then compared between HERDA-affected and unaffected horses using a Student's t-test. The modulus of skin from HERDA-affected horses decreased significantly more than that from unaffected horses following collagenase treatment (54 ± 7% versus 30 ± 16%, P = 0.004). The significant decrease in the modulus of skin from HERDA-affected horses following collagenase exposure suggests that their altered collagen microarchitecture is more susceptible to enzymatic degradation and may explain the localization of skin lesions in HERDA-affected horses to those areas of the body most exposed to sunlight. These findings appear to support the previously reported benefits of sunlight restriction in HERDA-affected horses.

Summary: Objectives: To determine if regional variations in strain patterns occur within the suspensory ligament under tensile load. Local increases in strain may put certain regions of the suspensory ligament at risk and may explain the poor healing and high recurrence rates associated with suspensory branch injuries.Methods: The suspensory ligament and its bone attachments were isolated from each of 10 adult equine cadaveric forelimbs and radiodense reference beads were inserted throughout the length of the ligament. Specimens were attached to a custom fixture secured to a materials testing system. Radio-graphs were acquired at 50, 445, 1112, and 2224 N of applied tensile load. Changes in distances between the beads in each region of the suspensory ligament were measured and the regional strain was calculated. Significant differences were determined using a repeated-measures analysis of variance.Results: The suspensory ligament exhibited significant differences in regional strain (p <0.001). The distal branches of the suspensory ligament had significantly greater strains than the proximal (p = 0.025) and mid-body (p = 0.002) regions. The mid-body of the suspensory ligament also exhibited local strain variation, with the distal mid-body having significantly higher strains than the proximal mid-body (p = 0.038).Clinical significance: The equine suspensory ligament demonstrates a heterogeneous strain pattern during tensile loading, with the distal regions exhibiting significantly more strain than the proximal region. The nonhomogenous strain pattern could explain the regional difference in injury and re-injury rates.

Background: Tendons are viscoelastic tissues that deform (elongate) in response to cyclic loading. However, the ability of a tendon to recover this elongation is unknown. Hypothesis: Tendon length significantly increases after in vivo or in vitro cyclic loading, and the ability to return to its original length through a cell-mediated contraction mechanism is an age-dependent phenomenon. Study Design: Controlled laboratory study. Methods: In vitro, rat tail tendon fascicles (RTTfs) from Sprague-Dawley rats of 3 age groups (1, 3, and 12 months) underwent 2% cyclic strain at 0.17 Hz for 2 hours, and the percentages of elongation were determined. After loading, the RTTfs were suspended for 3 days under tissue culture conditions and photographed daily to determine the amount of length contraction. In vivo, healthy male participants (n = 29; age, 19-49 years) had lateral, single-legged weightbearing radiographs taken of the knee at 60° of flexion immediately before, immediately after, and 24 hours after completing eccentric quadriceps loading exercises on the dominant leg to fatigue. Measurements of patellar tendon length were taken from the radiographs, and the percentages of tendon elongation and subsequent contraction were calculated. Results: In vitro, cyclic loading increased the length of all RTTfs, with specimens from younger (1 and 3 months) rats demonstrating significantly greater elongation than those from older (12 months) rats ( P = .009). The RTTfs contracted to their original length significantly faster ( P < .001) and in an age-dependent fashion, with younger animals contracting faster. In vivo, repetitive eccentric loading exercises significantly increased patellar tendon length ( P < .001). Patellar tendon length decreased 24 hours after exercises ( P < .001) but did not recover completely ( P < .001). There was a weak but significant ( R ² = 0.203, P = .014) linear correlation between the amount of tendon contraction and age, with younger participants (30 years). Conclusion: Cyclic tendon loading results in a significant increase in tendon elongation under both in vitro and in vivo conditions. Tendons in both conditions demonstrated an incomplete return to their original length after 24 hours, and the extent of this return was age dependent. Clinical Relevance: The age- and time-dependent contraction of tendons, elongated after repetitive loading, could result in transient alterations in the mechanobiological environment of tendon cells. This, in turn, could induce the onset of catabolic changes associated with the pathogenesis of tendinopathy. These results suggest the importance of allowing time for contraction between bouts of repetitive exercise and may explain why age is a predisposing factor in tendinopathy.

Summary: To describe the intra-osseous microvasculature of the distal phalanx of the equine forelimb with regard to its potential clinical relevance.Eleven clinically normal equine forelimbs were used from six adult horses (range: 4 to 18 years old) euthanatized for reasons unrelated to lameness. In each limb the median artery was catheterized at the level of the carpus and India ink was injected under constant manual pressure. The limbs were frozen and 5 mm thick sections of the foot were cut in the sagittal, coronal, or transverse planes on a band saw. The sections were fixed in 10% formalin and cleared using a modified Spalteholz technique. Once cleared, the sections were photographed and the microvascular anatomy identified.The vascular injections revealed a rich intra-osseous microvascular supply of the distal phalanx originating from the medial and lateral palmar digital arteries. In addition, numerous smaller vessels from the terminal arch, formed by anastomosis of the medial and lateral palmar digital arteries, could be seen branching into the distal aspects of the distal phalanx. This distal portion of the distal phalanx appeared more densely vascularized than the proximal part in all specimens examined.The increased vascularity demonstrated in the distal portion of the distal phalanx appears to correlate with improved fracture healing reported in this area. This may also explain why healing fractures which involve both the distal and proximal portions of the distal phalanx have been described as progressing from distal-to-proximal.

Reasons for performing study Studies examining the effect of stifle joint angle on tibial rotation, adduction–abduction angle and articular contact area are lacking. Objectives To test the hypothesis that tibial rotation, adduction–abduction angle and articular contact area change with stifle joint angle. Study design Descriptive study of normal kinematics and articular contact patterns of the equine stifle through the functional range of motion using 3 dimensional (3D) radiosteriometric analysis (RSA) and equine cadaver stifles. Methods Multiple, radiopaque markers were embedded in the distal femur and proximal tibia and sequential, biplanar x‐rays captured as the stifle was passively extended from 110° to full extension. Computer‐programmed RSA was used to determine changes in abduction–adduction and internal–external rotation angles of the tibia during stifle extension as well as articular contact patterns (total area and areas of high contact) through the range of motion. Results The tibia rotated externally (P<0.001) as the stifle was extended. Tibial abduction occurred from 110–135° of extension (P<0.001) and tibial adduction occurred from 135° through full extension (P = 0.009). The centre of joint contact moved cranially on both tibial condyles during extension with the lateral moving a greater distance than the medial (P = 0.003). Articular contact area decreased (P = 0.001) in the medial compartment but not in the lateral compartment (P = 0.285) as the stifle was extended. The area of highest joint contact increased on the lateral tibial condyle (P<0.001) with extension but decreased (P = 0.001) on the medial tibial condyle. Conclusions Significant changes occur in tibial rotation, adduction–abduction angle and articular contact area of the equine stifle through the functional range of motion. Understanding the normal kinematics of the equine stifle and the relationship between joint positions and articular contact areas may provide important insight into the aetiology and location of common stifle joint pathologies (articular cartilage and meniscal lesions).

Objective—To describe the effect of systemically administered oxytetracycline on the viscoelastic properties of rat tail tendon fascicles (TTfs) to provide a mechanistic rationale for pharmacological treatment of flexural limb deformities in foals. Sample—TTfs from ten 1-month-old and ten 6-month-old male Sprague-Dawley rats. Procedures—5 rats in each age group were administered oxytetracycline (50 mg/kg, IP, q 24 h) for 4 days. The remaining 5 rats in each age group served as untreated controls. Five days after initiation of oxytetracycline treatment, TTfs were collected and their viscoelastic properties were evaluated via a stress-relaxation protocol. Maximum modulus and equilibrium modulus were compared via a 2-way ANOVA. Collagen fibril size, density, and orientation in TTfs were compared between treated and control rats. Results—Viscoelastic properties were significantly decreased in TTfs from 1-month-old oxytetracycline-treated rats, compared with those in TTfs from 1-month-old control rats. Oxytetracycline had no effect on the viscoelastic properties of TTfs from 6-month-old rats. Collagen fibril size, density, and orientation in TTfs from 1-month-old rats did not differ between oxytetracycline-treated and control rats. Conclusions and Clinical Relevance—Results confirmed that systemically administered oxytetracycline decreased the viscoelastic properties of TTfs from 1-month-old rats but not those of TTfs from 6-month-old rats. The decrease in viscoelastic properties associated with oxytetracycline treatment does not appear to be caused by altered collagen fibril diameter or organization. The age-dependent effect of oxytetracycline on the viscoelastic properties of tendons may be related to its effect on the maturation of the extracellular matrix of developing tendons.