Objectives. This study aimed to evaluate the histological and mechanical features of tendon healing in a rabbit model with second-harmonic-generation (SHG) imaging and tensile testing. Materials and Methods. A total of eight male Japanese white rabbits were used for this study. The flexor digitorum tendons in their right leg were sharply transected, and then were repaired by intratendinous stitching. At four weeks post-operatively, the rabbits were killed and the flexor digitorum tendons in both right and left legs were excised and used as specimens for tendon healing (n = 8) and control (n = 8), respectively. Each specimen was examined by SHG imaging, followed by tensile testing, and the results of the two testing modalities were assessed for correlation. Results. While the SHG light intensity of the healing tendon samples was significantly lower than that of the uninjured tendon samples, 2D Fourier transform SHG images showed a clear difference in collagen fibre structure between the uninjured and the healing samples, and among the healing samples. The mean intensity of the SHG image showed a moderate correlation (R. 2. = 0.37) with Young’s modulus obtained from the tensile testing. Conclusion. Our results indicate that SHG microscopy may be a potential indicator of tendon healing. Cite this article: E. Hase, K. Sato, D. Yonekura, T. Minamikawa, M. Takahashi, T. Yasui. Evaluation of the histological and mechanical features of tendon healing in a rabbit model with the use of second-harmonic-generation imaging and tensile testing. Bone Joint Res 2016;5:577–585. DOI: 10.1302/2046-3758.511.BJR-2016-0162.R1

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1β, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1β, PDGF-BB, and TGF-β1 in fibroblasts infiltrating the frozen-thawed patellar tendon. Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1β than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1β was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-β1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1β had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons. The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment

Little is known about the increase in length of tendons in postnatal life or of their response to limb lengthening procedures. A study was carried out in ten young and nine adult rabbits in which the tibia was lengthened by 20% at two rates 0.8 mm/day and 1.6 mm/day. The tendon of the flexor digitorum longus (FDL) muscle showed a significant increase in length in response to lengthening of the tibia. The young rabbits exhibited a significantly higher increase in length in the FDL tendon compared with the adults. There was no difference in the amount of lengthening of the FDL tendon at the different rates. Of the increase in length which occurred, 77% was in the proximal half of the tendon. This investigation demonstrated that tendons have the ability to lengthen during limb distraction. This occurred to a greater extent in the young who showed a higher proliferative response, suggesting that there may be less need for formal tendon lengthening in young children

We split 100 porcine flexor tendons into five groups of 20 tendons for repair. Three groups were repaired using the Pennington modified Kessler technique, the cruciate or the Savage technique, one using one new device per tendon and the other with two new devices per tendon. Half of the tendons received supplemental circumferential Silfverskiöld type B cross-stitch. The repairs were loaded to failure and a record made of their bulk, the force required to produce a 3 mm gap, the maximum force applied before failure and the stiffness. When only one device was used repairs were equivalent to the Pennington modified Kessler for all parameters except the force to produce a 3 mm gap when supplemented with a circumferential repair, which was equivalent to the cruciate. When two devices were used the repair strength was equivalent to the cruciate repair, and when the two-device repair was supplemented with a circumferential suture the force to produce a 3 mm gap was equivalent to that of the Savage six-strand technique

We investigated the effect of mitomycin-C on the reduction of the formation of peritendinous fibrous adhesions after tendon repair. In 20 Wistar albino rats the tendo Achillis was cut and repaired using a modified Kessler technique. The rats were divided into two equal groups. In group 1, an injection of mitomycin-C was placed between the tendon and skin of the right leg. In group 2, an identical volume of sterile normal saline was injected on the left side in a similar fashion. All the rats received mitomycin-C or saline for four weeks starting from the day of operation. The animals were killed after 30 days. The formation of peritendinous fibrous tissue, the inflammatory reaction and tendon healing were evaluated. The tensile strength of the repaired tendons was measured biomechanically. Microscopic evidence of the formation of adhesions and inflammation was less in group 1. There was no significant difference in the tensile load required to rupture the repaired tendons in the two groups. Mitomycin-C may therefore provide a simple and inexpensive means of preventing of post-operative adhesions

The purpose of this anatomical study was to explore the morphological variations of the semitendinosus and gracilis tendons in length and cross-section and the statistical relationship between length, cross-section, and body height. We studied the legs of 93 humans in 136 cadavers. In 43 specimens (46.2%) it was possible to harvest the tendons from both legs. We found considerable differences in the length and cross-section of the semitendinosus and the gracilis tendons with a significant correlation between the two. A correlation between the length of the femur, reflecting height, and the length of the tendons was only observed in specimens harvested from women. The reason for this gender difference was unclear. Additionally, there was a correlation between the cross-sectional area of the tendons and the length of the femur. Surgeons should be aware of the possibility of encountering insufficient length of tendon when undertaking reconstructive surgery as a result of anatomical variations between patients

Operative fixation is the treatment of choice for a rupture of the distal tendon of biceps. A variety of techniques have been described including transosseous sutures and suture anchors. The poor quality of the bone of the radial tuberosity might affect the load to failure of the tendon repair in early rehabilitation. The aim of this study was to determine the loads to failure of different techniques of fixation and to investigate their association with the bone mineral density of the radial tuberosity. Peripheral quantitative computed tomography was carried out to measure the trabecular and cortical bone mineral density of the radial tuberosity in 40 cadaver specimens. The loads to failure in four different techniques of fixation were determined. The Endobutton-based method showed the highest failure load at 270 N (. sd. 22) (p < 0.05). The mean failure load of the transosseous suture technique was 210 N (. sd. 66) and that of the TwinFix-QuickT 5.0 mm was 57 N (. sd. 22), significantly lower than those of all other repairs (p < 0.05). No significant correlation was seen between bone mineral density and loads to failure. The transosseous technique is an easy and cost-saving procedure for fixation of the distal biceps tendon. TwinFix-QuickT 5.0 mm had significantly lower failure loads, which might affect early rehabilitation, particularly in older patients

We carried out lacerations of 50%, followed by trimming, in ten turkey flexor tendons in vitro and measured the coefficient of friction at the tendon-pulley interface with loads of 200 g and 400 g and in 10°, 30°, 50° and 70° of flexion. Laceration increased the coefficient of friction from 0.12 for the intact tendon to 0.3 at both the test loads. Trimming the laceration reduced the coefficient of friction to 0.2. An exponential increase in the gliding resistance was found at 50° and 70° of flexion (p = 0.02 and p = 0.003, respectively) following trimming compared to that of the intact tendon. We concluded that trimming partially lacerated flexor tendons will reduce the gliding resistance at the tendon-pulley interface, but will lead to fragmentation and triggering of the tendon at higher degrees of flexion and loading. We recommend that higher degrees of flexion be avoided during early post-operative rehabilitation following trimming of a flexor tendon

Objectives . The effects of disease progression and common tendinopathy treatments on the tissue characteristics of human rotator cuff tendons have not previously been evaluated in detail owing to a lack of suitable sampling techniques. This study evaluated the structural characteristics of torn human supraspinatus tendons across the full disease spectrum, and the short-term effects of subacromial corticosteroid injections (SCIs) and subacromial decompression (SAD) surgery on these structural characteristics. . Methods . Samples were collected inter-operatively from supraspinatus tendons containing small, medium, large and massive full thickness tears (n = 33). Using a novel minimally invasive biopsy technique, paired samples were also collected from supraspinatus tendons containing partial thickness tears either before and seven weeks after subacromial SCI (n = 11), or before and seven weeks after SAD surgery (n = 14). Macroscopically normal subscapularis tendons of older patients (n = 5, mean age = 74.6 years) and supraspinatus tendons of younger patients (n = 16, mean age = 23.3) served as controls. Ultra- and micro-structural characteristics were assessed using atomic force microscopy and polarised light microscopy respectively. . Results. Significant structural differences existed between torn and control groups. Differences were identifiable early in the disease spectrum, and increased with increasing tear size. Neither SCI nor SAD surgery altered the structural properties of partially torn tendons seven weeks after treatment. . Conclusions . These findings may suggest the need for early clinical intervention strategies for torn rotator cuff tendons in order to prevent further degeneration of the tissue as tear size increases. Further work is required to establish the long-term abilities of SCI and SAD to prevent, and even reverse, such degeneration. Cite this article: Bone Joint Res 2014;3:252–61

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

We studied the vascular pattern of human posterior tibial tendons by injection techniques and immunohistochemically using antibodies against laminin. The intravascular volume of the posterior tibial tendon was determined using a new method of injection of a solution of . 99m. Tc and gelatin ink into the lower legs of cadavers. Three segments of 1 cm length from different regions of the human posterior tibial tendon were measured using a gamma well counter. The main blood supply arises from the posterior tibial artery. Blood vessels enter the paratenon of the posterior tibial tendon via a mesotenon from the posterior aspect. From the paratenon, the blood vessels penetrate the posterior tibial tendon and anastomose with a longitudinally orientated intratendinous network. The number of vessels in the substance of the tendon is consistently less than that in the surrounding paratenon. The distribution of blood vessels within the posterior tibial tendon is not homogeneous. In the retromalleolar region the intravascular volume was significantly reduced with a mean value of 15 μl/g of tendon tissue. There was no significant difference between the mean intravascular volumes of the proximal and distal areas (distal, 27.7 μl/g tendon tissue; proximal, 30 μl/g tendon tissue). The immunohistochemical investigation showed that there was no immunostaining for laminin in the anterior part of the tendon in the region where it passes behind the medial malleolus. This region is avascular. The most frequent site of rupture of the posterior tibial tendon is in the region behind the medial malleolus. A potential endogenous risk factor may be the limited healing potential of avascular tissue

We performed a biomechanical and histological study to clarify the effect of stress enhancement on the in situ frozen-thawed patellar tendon of the rabbit as a tendon autograft model. We used 48 Japanese White rabbits divided into three groups. In group 1, the patellar tendon underwent in situ freeze-thaw treatment with liquid nitrogen to kill intrinsic fibroblasts. In group 2, after similar treatment, the medial and lateral portions were resected so that the cross-sectional area was reduced by a third. In group 3, after treatment, the cross-sectional area was reduced by a half. In groups 2 and 3, the stress in the tendon was calculated theoretically to be 150% and 200% of the physiological stress during locomotion. Eight rabbits in each group were killed at three and six weeks, respectively. At three weeks, the mean values for the tensile strength of groups 2 and 3 were 113.7% and 75.7% of that of group 1, and at six weeks 101.2% and 57.4%, respectively. The tensile strength in group 3 was significantly lower than that in groups 1 and 2. The histological findings in group 2 were similar to those in group 1, although an acellular area appeared to be wider in the core portion compared with group 1 at each period. In group 3, the collagen bundles of the tendon were less organised than those of groups 1 and 2. Our findings showed that stress enhancement affects the remodelling of the frozen-thawed patellar tendon and that excessively high stress reduces the mechanical properties of the tendon. This indicates that high stress on the patellar tendon autograft should be avoided during ligament reconstruction

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure. Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres

We released the infraspinatus tendons of six sheep, allowed retraction of the musculotendinous unit over a period of 40 weeks and then performed a repair. We studied retraction of the musculotendinous unit 35 weeks later using CT, MRI and macroscopic dissection. The tendon was retracted by a mean of 4.7 cm (3.8 to 5.1) 40 weeks after release and remained at a mean of 4.2 cm (3.3 to 4.7) 35 weeks after the repair. Retraction of the muscle was only a mean of 2.7 cm (2.0 to 3.3) and 1.7 cm (1.1 to 2.2) respectively at these two points. Thus, the musculotendinous junction had shifted distally by a mean of 2.5 cm (2.0 to 2.8) relative to the tendon. Sheep muscle showed an ability to compensate for approximately 60% of the tendon retraction in a hitherto unknown fashion. Such retraction may not be a quantitatively reliable indicator of retraction of the muscle and may overestimate the need for elongation of the musculotendinous unit during repair

We compared the biological characteristics of extrinsic fibroblasts infiltrating the patellar tendon with those of normal, intrinsic fibroblasts in the normal tendon in vitro. Infiltrative fibroblasts were isolated from the patellar tendons of rabbits six weeks after an in situ freeze-thaw treatment which killed the intrinsic fibroblasts. These intrinsic cells were also isolated from the patellar tendons of rabbits which had not been so treated. Proliferation and invasive migration into the patellar tendon was significantly slower for infiltrative fibroblasts than for normal tendon fibroblasts. Flow-cytometric analysis indicated that expression of α5β1 integrin at the cell surface was significantly lower in infiltrative fibroblasts than in normal tendon fibroblasts. The findings suggest that cellular proliferation and invasive migration of fibroblasts into the patellar tendon after necrosis are inferior to those of the normal fibroblasts. The inferior intrinsic properties of infiltrative fibroblasts may contribute to a slow remodelling process in the grafted tendon after ligament reconstruction

Despite widespread use of radiofrequency (RF) shrinkage, there have been no animal studies on the effects of post-operative immobilisation on the histological properties of the shrunken tissue. We have therefore examined the role of post-operative immobilisation after RF shrinkage with special emphasis on the histological properties of collagenous tissue. One patellar tendon of 66 New Zealand White rabbits was shrunk. Six rabbits were killed immediately after the operation. Twenty rabbits were not immobilised, 20 were immobilised for three weeks and 20 for six weeks. Fibroblasts, collagen and vascular quality and density were evaluated on sections, stained by haematoxylin and eosin. Nine weeks after operation the histological properties were inferior to those of the contralateral control tendons. Shrunk tendons did not return to normal at any time after operation irrespective of whether the animals had been immobilised or not. All the parameters improved significantly between zero and three weeks after operation. Immobilised tendons tended to have a better and faster recovery. Careful rehabilitation is imperative after RF shrinkage. Immobilisation aids recovery of the histological properties. Our findings in this animal model support a period of immobilisation of more than three weeks

We examined macroscopically and microscopically 55 cadaver rotator-cuff tendons attached to their humeral heads to determine the distance between the edge of the articular cartilage and the tendon insertion of the supraspinatus (the width of the sulcus) and the score of regressive changes at the sulcus. In 33 specimens we measured the tensile strength. The width of the sulcus was correlated with the score of regressive changes and with the ultimate tensile strength of the supraspinatus tendon. The width of the sulcus correlated positively with the score of regressive changes (r = 0.66, p < 0.0001), but there was a negative correlation between the latter and the ultimate tensile strength (r = −0.81, p = 0.001) and between the width of the sulcus and the ultimate tensile strength (r = −0.74, p = 0.004). We believe that the width of the sulcus is a simple and useful clinical indicator of the integrity and the tensile strength of the supraspinatus tendon

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres

In 14 rabbits we determined the origin of the cells effecting healing of the tendon of supraspinatus inserted into a bony trough. After two weeks both the cellularity of the underlying bone and the thickness of the subacromial bursa were significantly increased in the operated compared with the control shoulders. The cellularity of the stump of the tendon, however, was significantly decreased in the operated shoulders. In this model, both the underlying bone and the subacromial bursa but not the stump of the tendon contributed to the process of repair. We conclude that the medial stump should be debrided judiciously but that cutting back to bleeding tissue is not necessary during repair of the rotator cuff. Moreover, great care should be taken to preserve the subacromial bursa since it seems to play an important role in the healing process