Growth factor and protease expression during different phases of healing after rabbit deep flexor tendon repair

The purpose of the study was to contribute to the mapping of molecular events during flexor tendon healing, in particular the growth factors insulin‐like growth factor‐1 (IGF‐1), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), matrix metalloproteinases (MMP‐3 and MMP‐13) and their inhibitors (tissue inhibitors of metalloproteinases, TIMP‐1 and TIMP‐3, and the protease cathepsin K. In a rabbit model of flexor tendon injury, the mRNA expression for the growth factors, MMPs and TIMPs were measured in tendon and tendon sheath tissue at several time points (3, 6, 21, and 42 days) representing different phases of the healing process. We found that MMP‐13 remained increased during the study period, whereas MMP‐3 returned to normal levels within the first week after injury. TIMP‐3 was down‐regulated in the tendon sheaths. Cathepsin K was up‐regulated in tendons and sheaths after injury. NGF was present in both tendons and sheaths, but unaltered. IGF‐1 exhibited a late increase in the tendons, while VEGF was down‐regulated at the later time points. In conclusion, we have demonstrated the presence of NGF in flexor tendons. MMP‐13 expression appears to play a more protracted role in flexor tendon healing than MMP‐3. The relatively low levels of endogenous IGF‐1 and VEGF mRNA following injury support their potential beneficial role as exogenous modulators to optimize tendon healing and strength without increasing adhesion formation. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:886–892     

Microarray analysis of healing rat Achilles tendon: evidence for glutamate signaling mechanisms and embryonic gene expression in healing tendon tissue.

Tendon healing is a complex process consisting of a large number of intricate pathways roughly divided into the phases of inflammation, proliferation, and remodeling. Although these processes have been extensively studied at a variety of levels in recent years, there is still much that remains unknown. This study used microarray analyses to investigate the process at a genetic level in healing rat Achilles tendon at 1, 7, and 21 days postinjury, roughly representing the inflammation, proliferation, and remodeling phases. An interesting temporal expression profile was demonstrated, identifying both known and novel genes and pathways involved in the progression of tendon healing. Both inflammatory response and pro-proliferative genes were shown to be significantly upregulated from 24 h postinjury through to 21 days. Day 7 showed the largest increase in genetic activity, particularly with the expression of collagens and other extracellular matrix genes. Interestingly, there was also evidence of central nervous system-like glutamate-based signaling machinery present in tendon cells, as has recently been shown in bone. This type of signaling mechanism has not previously been shown to exist in tendon. Another novel finding from these analyses is that there appears to be several genes upregulated during healing which have exclusively or primarily been characterized as key modulators of proliferation and patterning during embryonic development. This may suggest that similar pathways are employed in wound healing as in the tightly regulated progression of growth and development in the embryo. These results could be of use in designing novel gene-based therapies to increase the efficacy and efficiency of tendon healing.     

CD44 deficiency improves healing tendon mechanics and increases matrix and cytokine expression in a mouse patellar tendon injury model

CD44 plays an important role in inflammation and healing. Previous studies investigated its role in inflammatory diseases and skin wounds; however, the role of CD44 in tendon healing is unknown. Therefore, we investigated the effect of CD44 in the healing of the patellar tendon in a knockout mouse model. We hypothesized that in comparison to wild‐type counterparts, CD44 knockout mice would have decreased material parameters, increased organization, decreased expression of proinflammatory cytokines, and increased expression of matrix components during healing. These hypotheses were tested through an in vivo surgical model and mechanical, organizational, and gene expression analyses. Material strength and tissue organization were significantly improved in the CD44 knockout mouse. This could be attributed to increased expression of cytokines and matrix components that are also elevated in regenerative healing. Our study showed that the absence of CD44 in a mouse patellar tendon injury creates an environment that is conducive to regenerative healing through altered gene expression, resulting in superior material properties and reduced cross‐sectional area. Therefore, limiting the role of CD44 may improve healing parameters in adult tendon injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1386–1391, 2009     

Varus knee osteoarthritis: Elevated synovial CD15 counts correlate with inferior biomechanical properties of lateral‐compartment cartilage

The study analyzed the influence of synovitis on the histological and biomechanical properties of lateral‐compartment cartilage. In a prospective cohort study, 84 patients (100 knees) with varus deformity of the knee were included. Osteochondral samples from the distal lateral femur underwent biomechanical and histologic analysis. Synovial tissue was sampled for histological (chronic synovitis score) and immunohistochemical evaluation of the degree of synovitis. CD15 (neutrophils), Ki‐67 (dividing cells), and CD68 (macrophages) were tested in all synovial samples. While the histological synovitis score did not correlate with the degree of cartilage degeneration (histological OARSI grades), both CD15 (r_s = 0.297, p = 0.006) and Ki‐67 (r_s = 0.249, p = 0.023) correlated with histological OARSI grades. There was a weak negative correlation of CD15 with biomechanical properties of cartilage of the distal lateral femur (aggregate modulus (Ha): r_s = ?0.125; p = 0.257; dynamic modulus (DM): r_s = ?0.216; p = 0.048). No correlations were observed for Ki‐67 and CD68. In addition, biomechanical properties were inferior in knees with a CD15 of >8/high power field compared to knees with a CD15 of ≤8/high power field (Ha: p = 0.031, d = 0.46; DM: p = 0.005, d = 0.68). The study demonstrates an association of increased inflammatory activity with advanced cartilage degeneration. Lateral‐compartment cartilage in knees with elevated synovial CD15 counts has a reduced ability to withstand compressive loads. CD15 might serve as an indicator for inferior biomechanical cartilage properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:841–846, 2018.

     

Gender‐specific in vivo measurement of the structural and mechanical properties of the human patellar tendon

Human patellar tendon stress (σ), strain (ε), stiffness (K), and tensile or Young's modulus (E), are determined in vivo through voluntary isometric contractions monitored with B‐mode ultrasonography. The limitations in previous studies are: (1) they have generally not accounted for the fact that the distal attachment of the patellar tendon (the tibial tuberosity) also displaces; thus, they have underestimated ε (and, hence, injury risk) while overestimating K; (2) no gender effect has been studied despite the fact that females are seen to have higher incidences of tendon‐related injuries. The current investigation therefore aimed to determine the gender specific values of σ, ε, K, and E of the patellar tendon while also accounting for distal displacement of the patellar tendon. Healthy young males (aged 23.1 ± 1.3 years, n = 10) and females (aged 21.3 ±0.9 years, n = 10) were tested. The maximal ε of the young males was ～5–10% higher than that reported in earlier literature. Average female versus male values for ε, σ, K, and E, taken at the same force level as the males for comparison purposes, were respectively 10.6 ± 1.0 versus 9.0 ± 1.0%, 36.9 ± 1.4 versus 28.9 ± 0.9 MPa, 1053 ± 108 versus 1652 ± 216 N · mm^?1, and 0.61 ± 0.08 versus 0.68 ± 0.10 GPa (p < 0.05). There are gender differences in tendon structural and mechanical properties. The current methodology may be useful in a clinical context where early prediction of injury risk and/or monitoring of reconstructed tendon needs to be an accurate, objective, and reliable method if optimal functionality is to be achieved. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1635–1642, 2007     

The native cell population does not contribute to central‐third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon

Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon‐to‐bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central‐third patellar tendon (PT) defect site. To do this, we contrasted the histochemical and biomechanical properties of de‐cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post‐surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12, or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 638–644, 2013     

The relationships among spatiotemporal collagen gene expression,histology, and biomechanics following full‐length injury in the murine patellar tendon

Tendon injuries are major orthopedic problems that worsen as the population ages. Type‐I (Col1) and type‐II (Col2) collagens play important roles in tendon midsubstance and tendon‐to‐bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full‐length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:28–36, 2012     

Efficient decellularization of equine tendon with preserved biomechanical properties and cytocompatibility for human tendon surgery indications

Chronic and acute tendon injuries are frequent afflictions, for which treatment is often long and unsatisfactory. When facing extended injuries, matrices and scaffolds with sufficient biomechanical properties are required for surgical repair and could additionally serve as supports for cellular therapies to improve healing. In this study, protocols of either commonly used detergents only (SDS 1%, Triton 1%, TBP 1%, and Tween-20 1%) or a combination of freeze/thaw (F/T) cycles with decellularization agents (NaCl 1M, ddH₂O) were evaluated for the decellularization of horse equine superficial digital flexor tendon (SDFT) for hand flexor or extensor tendon reconstruction. Decellularization efficiency was assessed microscopically by histological staining (HE, DAPI) and DNA quantification. Macroscopical structure and biomechanical integrity of the tendon matrices were further assessed by gross observation, histological staining (SR), and mechanical testing (ultimate strain and stress, Young’s modulus, energy to failure) for select protocols. Decellularization with hypertonic NaCl 1M in association with F/T cycles produced the most robust tendon matrices, which were nontoxic after 10 days for subsequent recellularization with human fetal progenitor tendon cells (hFPTs). This standardized protocol uses a less aggressive decellularization agent than current practice, which allows subsequent reseeding with allogenic cells, therefore making them very suitable and bioengineered tendon matrices for human tendon reconstruction in the clinic.     

Temporary inductions of matrix metalloprotease‐3 (MMP‐3) expression and cell apoptosis are associated with tendon degeneration or rupture after corticosteroid injection

Corticosteroid injections are widely used to treat enthesopathy and tendinitis, but are also associated with possible side effects, such as tendon degeneration or rupture. However, the mechanism of tendon degeneration or rupture after corticosteroid injection remains controversial. The purpose of this study was to reveal the mechanism of tendon degeneration or rupture after injection of triamcinolone acetonide (TA) or prednisolone (PSL). Forty‐two rats were divided into 3 groups: A normal saline injection group (control group), a TA injection group, and a PSL injection group; the normal saline or corticosteroid was injected around the Achilles tendon. One or 3 weeks after injection, the tendons were subjected to biomechanical testing and histological analysis. At 1 week, the biomechanical strength was significantly lower in the corticosteroid groups. Histological analysis, at 1‐week post‐injection, showed collagen attenuation, increased expression of MMP‐3 and apoptotic cells in the corticosteroid groups. The histological changes and biomechanical weaknesses of the tendon were not seen at 3 weeks. These alterations appeared to be involved in tendon degeneration or rupture after corticosteroid injection. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1297–1304, 2014.     

Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model

Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT‐PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK₁)‐ and calcitonin gene‐related peptide (CRLR and RAMP‐1)‐ receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to ～14× that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:274–280, 2009     

Sodium cromolyn reduces expression of CTGF,ADAMTS1, and TIMP3 and modulates post‐injury patellar tendon morphology

The purpose of this study was to determine whether administration of a mast cell inhibitor (sodium cromolyn, SC) would influence tendon repair and extracellular matrix gene expression following acute injury. CD1 mouse patellar tendons were unilaterally injured and mast cell prevalence was determined. The effect of SC injection on tendon hypercellularity, cross‐sectional area, collagen organization, and expression of extracellular matrix‐related genes was examined. Mast cell prevalence was markedly increased in injured patellar tendons (p = 0.009), especially at 8 weeks post‐injury (p = 0.025). SC injection increased collagen organization compared to uninjected animals at 4 weeks and attenuated the development of tendon hypercellularity and tendon thickening post‐injury. Expression of CTGF, ADAMTS1, and TIMP3 in injured tendon was reduced in the SC group. SC injections moderated the structural alterations of healing tendon in association with downregulation of several genes associated with tendon fibrosis. This work corroborates previous findings pointing to a role of mast cells in tendon repair. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:678–683, 2011     

Gait analysis combined with the expression of TGF-β1, TGF-β3 and CREB during Achilles tendon healing in rat

PurposeTo observe the changes of gait behavior and the expression of wound healing factors of transforming growth factor-β1 (TGF-β1), TGF-β3 and cAMP response element binding protein-1 (CREB-1) during the healing of Achilles tendon in a rat model, and to investigate whether gait analysis can be used to evaluate the tendon healing.MethodsAchilles tendon of 40 healthy male Sprague-Dawley rats were transected and sutured to establish the Achilles tendon injury (ATI) model. They were randomly divided into 4 groups based on the observational time point at 1, 2, 4 and 6 weeks after injury (n = 10 for each group). Before modeling, 9 rats were randomly selected for CatWalk gait analysis, which contained step cycle, single stance time and average speed. Data were recorded as the normal controls. After then, ATI models were established in the left hind limbs of the all 40 rats (ATI group), while the right hind limbs were only cut and sutured without injury of the Achilles tendon (sham operation group). At 1, 2, 4 and 6 weeks after injury, the gait behavior of the corresponding group of rats (n = 9) as observed and recorded by CatWalk platform. After then, the rats were sacrificed and Achilles tendon of both limbs was harvested. The tendon healing was observed by gross anatomy and histological examination, and the protein and mRNA expression of TGF-β1, TGF-β3, CREB-1 were observed by immunohistochemistry and qPCR. The results of tendon gross grading were analyzed by Wilcoxon rank sum test, and other data were analyzed by one-way analysis of variance among multiple groups.ResultsCompared with normal controls, all gait indexes (step cycle, single stance time and average speed) were greatly affected following ATI, which however improved with time. The step cycle was significantly lower at 1, 2 and 4 weeks after ATI (compared with normal controls, all p < 0.05), but almost returned to the normal level at 6 weeks ((0.694 ± 0.102) vs. (0.503 ± 0.094) s, p > 0.05). The single stance time of the ATI group was significantly shorter at 1 and 2 weeks after operation ((0.078 ± 0.010) s at 1 week, (0.078 ± 0.020) s at 2 weeks, all p < 0.001) and revealed no significant difference at 4 weeks (p = 0.120). The average speed of ATI group at 1, 2, 4, 6 weeks was significantly lower than that in the normal control group (all p < 0.001).Gross observation showed that the grade of local scar adhesion in ATI group increased significantly at 2, 4 and 6 weeks, compared with the sham operation group (all p < 0.001). Extensive adhesion was formed at 6 weeks after ATI. The results of HE staining showed that the number of fibroblast increased gradually and arranged more orderly in ATI group at 1, 2 and 4 weeks (all p < 0.001), and decreased at 6 weeks, but it was still significantly higher than that of the sham operation group (p < 0.001). Immunohistochemistry showed that the positive expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at 4 time points (all p < 0.05), which reached the peak at 2 weeks after operation and decreased at 4 weeks (p = 0.002, p < 0.001, p = 0.041, respectively). The results of qPCR suggested that the mRNA expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at all-time points (all p < 0.05), which reached the peak at 2 weeks after operation, decreased at 4 weeks, and significantly decreased at 6 weeks (all p < 0.001).ConclusionGait behavior indexes are associated with Achilles tendon healing. The study gives an insight of TGF-β1, TGF-β3, CREB-1 changes in the coursing of Achilles tendon healing and these cytokines may be able to be used to regulate the Achilles tendon healing.     

Tendon to bone tunnel healing—A study on the time‐dependent changes in biomechanics,bone remodeling,and histology in a rat model

Tendons and ligaments attach to bone through a transitional connective tissue with complex biomechanical properties. This unique tissue is not regenerated during healing, and surgical reattachment therefore often fails. The present study was designed to evaluate tendon healing in a bone tunnel and to evaluate the utilized rat model. Wistar rats (n = 61) were operated with the Achilles tendon through a bone tunnel in the distal tibia. Healing was evaluated at 2, 3, 4, and 12 weeks by biomechanical testing, bone mineral density and histology. After 2 weeks median (interquartile range) pull‐out force was 2.2 N (1.9). The pull‐out force increased chronologically, by 12 weeks fivefold to 11.2 N (11.4). Energy absorption, stiffness, and bone mineral density increased similarly. The histological analyses showed inflammation at early stages with increasing callus by time. Our data showed a slow healing response the first 4 weeks followed by an accelerated healing period, favoring that most of the gain in mechanical strength occurred later than 4 weeks postoperatively. These findings support the concern of a vulnerable tendon bone tunnel interface in the early stages of healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:216–223, 2015.     

Mechanical,compositional, and structural properties of the mouse patellar tendon with changes in biglycan gene expression

Tendons have complex mechanical properties that depend on their structure and composition. Some studies have assessed the role of small leucine‐rich proteoglycans (SLRPs) in the mechanical response of tendon, but the relationships between sophisticated mechanics, assembly of collagen and SLRPs have not been well characterized. In this study, biglycan gene expression was varied in a dose dependent manner using biglycan null, biglycan heterozygote and wild type mice. Measures of mechanical (tension and compression), compositional and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was found to be increased in the biglycan heterozygous and biglycan null tendons compared to wild type. Gene expression analyses revealed biglycan gene expression was closely associated in a dose‐dependent allelic manner. No differences were seen between genotypes in elastic or compressive properties or quantitative measures of collagen structure. These results suggest that biglycan, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1430–1437, 2013     

Combined application of low‐intensity pulsed ultrasound and functional electrical stimulation accelerates bone–tendon junction healing in a rabbit model

The objective of this study was to elucidate the combined use of low‐intensity pulsed ultrasound (LIPUS) and functional electrical stimulation (FES) on patella–patellar tendon (PPT) junction healing using a partial patellectomy model in rabbits. LIPUS was delivered continuously starting day 3 postoperative until week 6. FES was applied on quadriceps muscles to induce tensile force to the repaired PPT junction 5 days per week for 6 weeks since week 7 postoperatively. Forty rabbits with partial patellectomy were randomly divided into four groups: control, LIPUS alone, FES alone, and LIPUS + FES groups. At week 12, the PPT complexes were harvested for histology, radiographs, peripheral quantitative computed tomography, and biomechanical testing. There was better remodeling of newly formed bone and fibrocartilage zone in the three treatment groups compared with the control group. LIPUS and/or FES treatments significantly increased the area and bone mineral content of new bone. The failure load and ultimate strength of PPT complex were also highly improved in the three treatment groups. More new bone formed and higher tensile properties were showed in the LIPUS + FES group compared with the LIPUS or FES alone groups. Early LIPUS treatment and later FES treatment showed the additive effects of accelerating PPT junction healing. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:204–209, 2014.     

Indian hedgehog signaling and the role of graft tension in tendon‐to‐bone healing: Evaluation in a rat ACL reconstruction model

The structure and composition of the native enthesis is not recapitulated following tendon‐to‐bone repair. Indian Hedgehog (IHH) signaling has recently been shown to be important in enthesis development in a mouse model but no studies have evaluated IHH signaling in a healing model. Fourteen adult male rats underwent ACL reconstruction using a flexor tendon graft. Rats were assigned to two groups based on whether or not they received 0N or 10N of pre‐tension of the graft. Specimens were evaluated at 3 and 6 weeks post‐operatively using immunohistochemistry for three different protein markers of IHH signaling. Quantitative analysis of staining area and intensity using custom software demonstrated that IHH signaling was active in interface tissue formed at the healing tendon‐bone interface. We also found increased staining area and intensity of IHH signaling proteins at 3 weeks in animals that received a pre‐tensioned tendon graft. No significant differences were seen between the 3‐week and 6‐week time points. Our data suggests that the IHH signaling pathway is active during the tendon‐bone healing process and appears to be mechanosensitive, as pre‐tensioning of the graft at the time of surgery resulted in increased IHH signaling at three weeks. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:641–649, 2016.