Objectives. To compare the effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament (ACL) reconstruction animal model. Methods. Anterior cruciate ligament reconstruction using the plantaris tendon as graft material was performed on both knees of 24 rabbits (48 knees) to mimic ACL reconstruction by two different suspensory fixation devices for graft fixation. For the adjustable fixation device model (Socket group; group S), a 5 mm deep socket was created in the lateral femoral condyle (LFC) of the right knee. For the fixed-loop model (Tunnel group; group T), a femoral tunnel penetrating the LFC was created in the left knee. Animals were sacrificed at four and eight weeks after surgery for histological evaluation and biomechanical testing. Results. Histologically, both groups showed a mixture of direct and indirect healing patterns at four weeks, whereas only indirect healing patterns were observed in both groups at eight weeks. No significant histological differences were seen between the two groups at four and eight weeks in the roof zone (four weeks, S: mean 4.8 . sd. 1.7, T: mean 4.5 . sd. 0.5, p = 0.14; eight weeks, S: mean 5.8 . sd. 0.8, T: mean 4.8 . sd. 1.8, p = 0.88, Mann-Whitney U test) or side zone (four weeks, S: mean 5.0 . sd. 1.2, T: mean 4.8 . sd. 0.4, p = 0.43; eight weeks, S: mean 5.3 . sd. 0.8,T: mean 5.5 . sd. 0.8, p = 0.61, Mann-Whitney U test) . Similarly, no significant difference was seen in the maximum failure load between group S and group T at four (15.6 . sd. 9.0N and 13.1 . sd. 5.6N) or eight weeks (12.6 . sd. 3.6N and 17.1 . sd. 6.4N, respectively). Conclusion. Regardless of bone tunnel configuration, tendon-bone healing after ACL reconstruction primarily occurred through indirect healing. No significant histological or mechanical differences were observed between adjustable and fixed-loop femoral cortical suspension methods. Cite this article: Y. Sato, R. Akagi, Y. Akatsu, Y. Matsuura, S. Takahashi, S. Yamaguchi, T. Enomoto, R. Nakagawa, H. Hoshi, T. Sasaki, S. Kimura, Y. Ogawa, A. Sadamasu, S. Ohtori, T. Sasho. The effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament reconstruction: An animal study. Bone Joint Res 2018;7:327–335. DOI: 10.1302/2046-3758.75.BJR-2017-0238.R2

Aims

It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth.

Aims

To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction.

Introduction. It is an example of tendon to bone healing of rotator cuff tears. Low Level Laser Therapy (LLLT) is used in pain literature, pain palliation, tendinopathies, osteoarthritis treatment, implant osteointegration in jaw surgery, wound healing, fracture healing, tendon healing, nerve healing. But; there is not a study on tendon-bone healing. It is aimed to investigate the effects of tendon to bone healing with the rotator cuff experimental tear model. Material and Methods. 60 Wistor Albino Rat right shoulders were used in our project in four groups. Effectiveness of the study in each study group to increase and use the minimum number of animals that would be significant it was planned to use 15 (6 histology + 9 biomechanical) subjects. In our study, there are 4 groups in total. 1. Group 4. Week sacrified control group 2. Group 4. Week sacrificed LLLT group 3. Group 8. Week sacrified control group 4. Group is postoperative LLLT group. The 4 round SSP tendons have been cut with a full course. A total of eight sessions of biostimulation were performed with 24 j energy per session. Biomechanical tensile test and histopathological examination were performed on rats sacrificed at 4 and 8 weeks. In histological examination, cellularity at the repair site with hemotoxylin-eosin staining, extracellular matrix localization with Masson trichrome staining and fibrosis, TRAP (Tartrate Resistance Acid Phosphatase) and osteoclast activity, collagen fibril organization with picrochucine were evaluated. In immunohistological examination, proliferation activity was evaluated by CD-31 (Abcam, Cambridge, MA, USA) through vascular endothelial cells, Ki-67 (Tucson, AZ) and tendon proliferation index. Failure load for comparison of biomechanical stability between groups drawing will be done. Biomechanical pulling applications Linear pulling force over tendon It will be realized by applying. Last failure load (Newton), elongation (mm) and hardness values was recorded. Results. Two rats died in the postoperative first group and one rat died in the other groups. Six animals from each group were examined histopathologically. While the mean of stifness was higher in the patient who underwent LLLT at the 4th week biomechanically compared to the control group, there was no difference in the 8th week between the control group LLLT biomechanically. Histologically, the group with LLLT at 4 weeks showed increased fibroblastic activity and osteoclastic activity, but at 8 weeks there was no significant difference. Discussion. There are studies on the benefits of biostimulation with LLLT in fracture healing, implant integration, wound healing, tendon healing. Biostimulation experiments with LLLT for tendon healing were evaluated in rotator cuff healing as they were not available in literature. Especially early contributions were thought to be beneficial for postoperative rupture

Animal studies examining tendon-bone healing have demonstrated that the overall structure, composition, and organization of direct type entheses are not regenerated following repair. We examined the effect of Low-Intensity Pulsed Ultrasound (LIPUS) on tendon-bone healing. LIPUS may accelerate and augment the tendon-bone healing process through alteration of critical molecular expressions. Eight skeletally mature wethers, randomly allocated to either control group (n=4) or LIPUS group (n=4), underwent rotator cuff surgery following injury to the infraspinatus tendon. All animals were sacrificed 28 days post surgery to allow examination of early effects of LIPUS. Humeral head – infraspinatus tendon constructs were harvested and processed for histology and immunohistochemical staining for BMP2, Smad4, VEGF and RUNX2. All the growth factors were semiquantitative evaluated. T-tests were used to examine differences which were considered significant at p < 0.05. Levene's Test (p < 0.05) was used to confirm variance homogeneity of the populations. The surgery and LIPUS treatment were well tolerated by all animals. Placement of LIPUS sensor did not unsettle the animals. Histologic appearance at the tendon-bone interface in LIPUS treated group demonstrated general improvement in appearance compared to controls. Generally a thicker region of newly formed woven bone, morphologically resembling trabecular bone, was noted at the tendon-bone interface in the LIPUS-treated group compared to the controls. Structurally, treatment group also showed evidence of a mature interface between tendon and bone as indicated by alignment of collagen fibres as visualized under polarized light. Immunohistochemistry revealed an increase in the protein expression patterns of VEGF (p = 0.038), RUNX2 (p = 0.02) and Smad4 (p = 0.05) in the treatment group. There was no statistical difference found in the expression patterns of BMP2. VEGF was positively stained within osteoblasts in newly formed bone, endothelial cells and some fibroblasts at the interface and focally within fibroblasts around the newly formed vessels. Expression patterns of RUNX2 were similar to that of BMP-2; the staining was noted in active fibroblasts found at the interface as well as in osteoblast-like cells and osteoprogenitor cells. Immunostaining of Smad4 was present in all cell types at the healing interface. The results of this study indicate that LIPUS may aid in tendon to bone healing process in patients who have undergone rotator cuff repair. This treatment may also be beneficial following other types of reconstructive surgeries involving the tendon-bone interface

Background. Re-attachment of tendon to bone is challenging with surgical repair failing in up to 90% of cases. Poor biological healing is common and characterised by the formation of weak scar tissue. Previous work has demonstrated that decellularised allogenic demineralised bone matrix (DBM) regenerates a physiologic enthesis. Xenografts offer a more cost-effective option but concerns over their immunogenicity have been raised. We hypothesised that augmentation of a healing tendon-bone interface with DBM incorporated with autologous mesenchymal stem cells (MSCs) would result in improved function, and restoration of the native enthesis, with no difference between xenogenic and allogenic scaffolds. Methods. Using an ovine model of tendon-bone retraction the patellar tendon was detached and a complete distal tendon defect measuring 1 cm was created. Suture anchors were used to reattach the shortened tendon and xenogenic DBM + MSCs (n=5) and allogenic DBM + MSCs (n=5) were used to bridge the defect. Functional recovery was assessed every 3 weeks and DBM incorporation into the tendon and its effect on enthesis regeneration was measured using histomorphometry. Results. By 12 weeks, DBM augmentation resulted in significantly improved functional weight bearing with no failures in either group. Compared to xenogenic DBM, allogenic DBM was associated with significantly higher functional weight bearing at 6 (P=0.047), 9 (P=0.028) and 12 weeks (P=0.009). This was accompanied by a more direct type of enthesis characterised by significantly more fibrocartilage and mineralised fibrocartilage. Xenograft was also associated with an immunogenic reaction despite preoperative decellularisation. Conclusion. This study shows that DBM enhances tendon-bone healing and may reduce the high failure rates associated with surgery. An immunogenic reaction, and inferior biomechanical and histological results were also associated with the use of xenograft. Allogenic DBM with autologous MSCs may be a suitable scaffold for the enhancement of tendon-bone healing in the clinical setting. Disclosures. Funded by IKC PoC grant awarded by the University of Leeds. Ethical approval. Granted by the study institution (University College London)

Due to unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction new regenerative approaches based on tissue-engineering strategies are currently under investigation. It was the purpose of this study to determine if a novel silk fiber-based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions. Furthermore we tested if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell-rich isolate from knee fat pads. In this controlled laboratory study, 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro-computed tomography measurements were performed. The histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro-computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation. The novel silk fiber-based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed

An established rabbit model was used to preliminarily investigate the effect of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets on tendon-bone interface healing. Bone, fibrocartilage and tendon tissue were harvested from the rabbits and sectioned into a book-type scaffold. The scaffolds were decellularized and their characterization was presented. BMSCs were isolated and co-cultured with the scaffolds to verify their cytocompatibility. BMSCs sheets were fabricated and inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS complex. The complex was implated in the right knee of rabbits which operated standard partial patellectomy for TBI regeneration using Imaging, histological and biomechanical examinations. The bone, fibrocartilage and tendon tissue were sectioned into a book-type scaffold before decellularization. Then we decellularized the above tissue and mostly preserved their microstructure and composition of the natural extracellular matrix, including collagen and proteoglycan. After the physicochemical and biological properties of the book-type ATS were evaluated, autologous BMSCs sheets were inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS implants for TBI regeneration. In addition, the ATS has the advantages of non-toxicity, suitable for cell adhesion and growth as well as low immunogenicity while co-cultured with the BMSCs. At the same time, different scaffolds has the ability to induce the osteogenic, chondrogenic and tenogenic differentiation of BMSCs by immunofluorescence, reverse transcription-polymerase chain reaction and western blot analysis. To determine the efficacy of the tissue-engineered implants for TBI regeneration, we transplanted it into a rabbit patella-patellar tendon (PPT) injury model, and the rabbits were sacrificed at postoperative week 8 or 16 for the radiological, histological, and mechanical evaluation. Radiologically, Synchrotron radiation micro-computed tomography (SR-μCT) showed that BMSCs/ATS group significantly increased bone area, BV/TV, trabecular thickness and trabecular number at the healing interface as compared with other groups at postoperative week 8 or 16. Histologically, the BMSCs/ATS group showed more woven bone, and a more robust fibrocartilaginous junction with a characteristic matrix rich in proteoglycans was seen at the PPT healing interface in comparison with other groups after 8 weeks. At week 16, the healing interface in 3 groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT junction were almost complete, with a resemblance to a healthy BTI consisting of the characteristic 4 zones in all groups. At last, we used biomechanical test as functional parameters to evaluate the quality of tendon-bone healing. Biomechanical testing indicated that BMSCs/ATS group showed significantly higher failure load and stiffness than other groups at postoperative week 8 and 16. The complex composed of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets can simulate the gradient structure of tendon-bone interface, inducing stem cell directional differentiation, so as to promote patella-patellar tendon interface healing effectively after injury

Aims

Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing.

Fibrocartilaginous entheses are formed through endochondral ossification and characterized by four zones morphologically separated into tendon, uncalcified fibrocartilage, calcified fibrocartilage and bone [1]. These zones are not successfully regenerated following surgical repair. Demineralized Bone (DBM) presented at the tendon bone interface may improve healing between tendon and bone. Fifty six female nude rats were randomly allocated into either a control reconstruction or treatment group (DBM at the tendon-bone healing site). A modified rodent model of anterior cruciate ligament reconstruction was adopted [2]. Animals were sacrificed at 2, 4 and 6 weeks following surgery. Four rats per group were prepared for histology at each time point while eight rats were culled for biomechanical testing at 4 and 6 week time points. ANOVA and post hoc tests were used to examine differences which were considered significant at p < 0.05. The surgical procedure was well tolerated. Macroscopic dissection did not reveal any infection and all joint surfaces appeared normal. An intra-articular graft between the femur and tibia was present in all specimens. Mechanical differences were noted between groups. Peak loads were significantly higher in treatment group at 4 and 6 weeks (6.0 ± 3.6N and 9.1 ± 2.6 N, respectively) compared to controls (2.9 ± 1.9 N and 5.8 ± 2.7 N). No statistical differences were found in graft stiffness between the groups at 4 or 6 week time points. Histology showed an initial influx of inflammatory cells coupled with formation of a loose disorganized fibrovascular interface layer between tendon and bone in both groups. By the 6 weeks the interface layer in the DBM group fused into the newly formed bone to create a continuum between the tendon and bone, in an interdigitated fashion, containing Sharpy's like fibres. In the control group the continuum was less apparent with evidence of large areas of discontinuity between the two zones. A thicker region of newly formed woven bone with increased osteoblast activity along the bone tunnel was evident in the DBM group. DBM has the potential to increase the quality of repair following surgical procedures involving reattachment of tendon to bone

Research in to tendon-bone healing techniques focus on increasing bone growth at the interface such as cell or growth factor (e.g. BMP-2) augmentation. Demineralised bone matrix (DBM) is osseoinductive and is in use clinically. Hypothesis: DBM augmentation of a healing tendon-bone interface will result in improved function at 3, 6, 9 and 12 weeks, and a morphology that more closely resembles that of a normal enthesis at 12 weeks. Materials and methods: An ovine patellar tendon model was used. 19 skeletally mature ewes were allocated to the control group or DBM group. In both groups the patellar tendon was detached, and following tibial tubercle osteotomy, was re-attached using 3 suture anchors. In the DBM group a piece of DBM was placed between the tendon and bone. 2 animals were sacrificed at 6 weeks and 6 animals at 12 weeks. Animals underwent force plate analysis at 3, 6, 9 and 12 weeks. The tendon-bone interface length which was fibrous or fibrocartilaginous, and the area of fibrocartilage, mineralised fibrocartilage and new bone was quantified. Results: 3 control group animals (33%) failed within 6 weeks. None failed in the DBM group. The DBM group was significantly better than the control at all time points (p< 0.05). DBM produced a significantly more fibrocartilaginous enthesis than the control group (p< 0.05). Controls were significantly more fibrous than the DBM group (p< 0.05). DBM produced significantly more fibrocartilage (p< 0.05), and mineralised fibrocartilage (p< 0.05). Discussion: 33% of the control group failed within 6 weeks, whilst no failures were observed in the DBM group. DBM animals mobilised earlier and had significantly better function at all time points. Histologically, the DBM group showed a more mature direct type enthesis at earlier time points. Conclusion: DBM augmentation of a healing tendon-bone interface enhances functional and morphological recovery at earlier time points

Aims

The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction.

Aims

To evaluate graft healing of decellularized porcine superflexor tendon (pSFT) xenograft in an ovine anterior cruciate ligament (ACL) reconstruction model using two femoral fixation devices. Also, to determine if pSFT allows functional recovery of gait as compared with the preoperative measurements.

Aims

Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells.

Objectives

The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.

Objectives

Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics.

Objectives

Although many clinical and experimental investigations have shed light on muscle atrophy and intramuscular accumulation of fat after rotator cuff disruption, none have reported on their onset in the absence of muscle retraction.