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The Journal of Bone & Joint Surgery British Volume
Vol. 93-B, Issue 2 | Pages 237 - 244
1 Feb 2011
Berber O Amis AA Day AC

The purpose of this study was to assess the stability of a developmental pelvic reconstruction system which extends the concept of triangular osteosynthesis with fixation anterior to the lumbosacral pivot point. An unstable Tile type-C fracture, associated with a sacral transforaminal fracture, was created in synthetic pelves. The new concept was compared with three other constructs, including bilateral iliosacral screws, a tension band plate and a combined plate with screws. The pubic symphysis was plated in all cases. The pelvic ring was loaded to simulate single-stance posture in a cyclical manner until failure, defined as a displacement of 2 mm or 2°. The screws were the weakest construct, failing with a load of 50 N after 400 cycles, with maximal translation in the craniocaudal axis of 12 mm. A tension band plate resisted greater load but failure occurred at 100 N, with maximal rotational displacement around the mediolateral axis of 2.3°.

The combination of a plate and screws led to an improvement in stability at the 100 N load level, but rotational failure still occurred around the mediolateral axis. The pelvic reconstruction system was the most stable construct, with a maximal displacement of 2.1° of rotation around the mediolateral axis at a load of 500 N.


Bone & Joint Research
Vol. 11, Issue 7 | Pages 503 - 512
25 Jul 2022
Wu Y Shao Y Xie D Pan J Chen H Yao J Liang J Ke H Cai D Zeng C

Aims

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

Methods

In vitro: the mobility of the rat bone mesenchymal stem cells (BMSCs) treated with SLPI was evaluated by scratch assay. Then the expression levels of osteogenic differentiation-related genes were analyzed by real-time quantitative PCR (qPCR) to determine the osteogenic effect of SLPI on BMSCs. In vivo: a rat model of ACL reconstruction was used to verify the effect of SLPI on tendon-to-bone healing. All the animals of the SLPI group and the negative control (NC) group were euthanized for histological evaluation, micro-CT scanning, and biomechanical testing.


The Bone & Joint Journal
Vol. 103-B, Issue 2 | Pages 294 - 298
1 Feb 2021
Hadeed MM Prakash H Yarboro SR Weiss DB

Aims

The aim of this study was to determine the immediate post-fixation stability of a distal tibial fracture fixed with an intramedullary nail using a biomechanical model. This was used as a surrogate for immediate weight-bearing postoperatively. The goal was to help inform postoperative protocols.

Methods

A biomechanical model of distal metaphyseal tibial fractures was created using a fourth-generation composite bone model. Three fracture patterns were tested: spiral, oblique, and multifragmented. Each fracture extended to within 4 cm to 5 cm of the plafond. The models were nearly-anatomically reduced and stabilized with an intramedullary nail and three distal locking screws. Cyclic loading was performed to simulate normal gait. Loading was completed in compression at 3,000 N at 1 Hz for a total of 70,000 cycles. Displacement (shortening, coronal and sagittal angulation) was measured at regular intervals.


Bone & Joint Research
Vol. 6, Issue 4 | Pages 216 - 223
1 Apr 2017
Ang BFH Chen JY Yew AKS Chua SK Chou SM Chia SL Koh JSB Howe TS

Objectives

External fixators are the traditional fixation method of choice for contaminated open fractures. However, patient acceptance is low due to the high profile and therefore physical burden of the constructs. An externalised locking compression plate is a low profile alternative. However, the biomechanical differences have not been assessed. The objective of this study was to evaluate the axial and torsional stiffness of the externalised titanium locking compression plate (ET-LCP), the externalised stainless steel locking compression plate (ESS-LCP) and the unilateral external fixator (UEF).

Methods

A fracture gap model was created to simulate comminuted mid-shaft tibia fractures using synthetic composite bones. Fifteen constructs were stabilised with ET-LCP, ESS-LCP or UEF (five constructs each). The constructs were loaded under both axial and torsional directions to determine construct stiffness.


The Journal of Bone & Joint Surgery British Volume
Vol. 94-B, Issue 5 | Pages 713 - 718
1 May 2012
Kaiser MM Zachert G Wendlandt R Eggert R Stratmann C Gros N Schulze-Hessing M Rapp M

Elastic stable intramedullary nailing (ESIN) is generally acknowledged to be the treatment of choice for displaced diaphyseal femoral fractures in children over the age of three years, although complication rates of up to 50% are described. Pre-bending the nails is recommended, but there are no published data to support this. Using synthetic bones and a standardised simulated fracture, we performed biomechanical testing to determine the influence on the stability of the fracture of pre-bending the nails before implantation. Standard ESIN was performed on 24 synthetic femoral models with a spiral fracture. In eight cases the nails were inserted without any pre-bending, in a further eight cases they were pre-bent to 30° and in the last group of eight cases they were pre-bent to 60°. Mechanical testing revealed that pre-bending to 60° produced a significant increase in the stiffness or stability of the fracture. Pre-bending to 60° showed a significant positive influence on the stiffness compared with unbent nails. Pre-bending to 30° improved stiffness only slightly.

These findings validate the recommendations for pre-bending, but the degree of pre-bend should exceed 30°. Adopting higher degrees of pre-bending should improve stability in spiral fractures and reduce the complications of varus deformity and shortening.


Bone & Joint Research
Vol. 12, Issue 12 | Pages 722 - 733
6 Dec 2023
Fu T Chen W Wang Y Chang C Lin T Wong C

Aims. Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration. Methods. A total of 24 male New Zealand white rabbits were divided into four groups according to the experimental materials. Allogenic adipose-derived mesenchymal stem cells (AMSCs) were cultured and seeded evenly in the collagen/chitosan sheet to form cell sheet as periosteum. Simultaneously, allogenic AMSCs were seeded onto alginate beads and were cultured to differentiate to endothelial-like cells to form vascularized bone construct (VBC). The cell sheet was wrapped onto VBC to create a vascularized bone-periosteum construct (VBPC). Four different experimental materials – acellular construct, VBC, non-vascularized bone-periosteum construct, and VBPC – were then implanted in bilateral L4-L5 intertransverse space. At 12 weeks post-surgery, the bone-forming capacities were determined by CT, biomechanical testing, histology, and immunohistochemistry staining analyses. Results. At 12 weeks, the VBPC group significantly increased new bone formation volume compared with the other groups. Biomechanical testing demonstrated higher torque strength in the VBPC group. Notably, the haematoxylin and eosin, Masson’s trichrome, and immunohistochemistry-stained histological results revealed that VBPC promoted neovascularization and new bone formation in the spine fusion areas. Conclusion. The tissue-engineered VBPC showed great capability in promoting angiogenesis and osteogenesis in vivo. It may provide a novel approach to create a superior blood supply and nutritional environment to overcome the deficits of current artificial bone graft substitutes. Cite this article: Bone Joint Res 2023;12(12):722–733


The Journal of Bone & Joint Surgery British Volume
Vol. 93-B, Issue 7 | Pages 937 - 941
1 Jul 2011
Bae J Oh J Chon C Oh C Hwang J Yoon Y

We evaluated the biomechanical properties of two different methods of fixation for unstable fractures of the proximal humerus. Biomechanical testing of the two groups, locking plate alone (LP), and locking plate with a fibular strut graft (LPSG), was performed using seven pairs of human cadaveric humeri. Cyclical loads between 10 N and 80 N at 5 Hz were applied for 1 000 000 cycles. Immediately after cycling, an increasing axial load was applied at a rate of displacement of 5 mm/min. The displacement of the construct, maximum failure load, stiffness and mode of failure were compared. The displacement was significantly less in the LPSG group than in the LP group (p = 0.031). All maximum failure loads and measures of stiffness in the LPSG group were significantly higher than those in the LP group (p = 0.024 and p = 0.035, respectively). In the LP group, varus collapse and plate bending were seen. In the LPSG group, the humeral head cut out and the fibular strut grafts fractured. No broken plates or screws were seen in either group. We conclude that strut graft augmentation significantly increases both the maximum failure load and the initial stiffness of this construct compared with a locking plate alone


Objectives. Secondary fracture healing is strongly influenced by the stiffness of the bone-fixator system. Biomechanical tests are extensively used to investigate stiffness and strength of fixation devices. The stiffness values reported in the literature for locked plating, however, vary by three orders of magnitude. The aim of this study was to examine the influence that the method of restraint and load application has on the stiffness produced, the strain distribution within the bone, and the stresses in the implant for locking plate constructs. Methods. Synthetic composite bones were used to evaluate experimentally the influence of four different methods of loading and restraining specimens, all used in recent previous studies. Two plate types and three screw arrangements were also evaluated for each loading scenario. Computational models were also developed and validated using the experimental tests. Results. The method of loading was found to affect the gap stiffness strongly (by up to six times) but also the magnitude of the plate stress and the location and magnitude of strains at the bone-screw interface. Conclusions. This study demonstrates that the method of loading is responsible for much of the difference in reported stiffness values in the literature. It also shows that previous contradictory findings, such as the influence of working length and very large differences in failure loads, can be readily explained by the choice of loading condition. Cite this article: A. MacLeod, A. H. R. W. Simpson, P. Pankaj. Experimental and numerical investigation into the influence of loading conditions in biomechanical testing of locking plate fracture fixation devices. Bone Joint Res 2018;7:111–120. DOI: 10.1302/2046-3758.71.BJR-2017-0074.R2


The Journal of Bone & Joint Surgery British Volume
Vol. 90-B, Issue 10 | Pages 1386 - 1391
1 Oct 2008
Ozbaydar M Elhassan B Esenyel C Atalar A Bozdag E Sunbuloglu E Kopuz N Demirhan M

We compared time-dependent changes in the biomechanical properties of single-and double-row repair of a simulated acute tear of the rotator cuff in rabbits to determine the effect of the fixation techniques on the healing process. A tear of the supraspinatus tendon was created in 80 rabbits which were separated into two equal groups. A single-row repair with two suture anchors was conducted in group 1 and a double-row repair with four suture anchors in group 2. A total of ten intact contralateral shoulder joints was used as a control group. Biomechanical testing was performed immediately post-operatively and at four and eight weeks, and histological analysis at four and eight weeks. The mean load to failure in group 2 animals was greater than in group 1, but both groups remained lower than the control group at all intervals. Histological analysis showed similar healing properties at four and eight weeks in both groups, but a significantly larger number of healed tendon-bone interfaces were identified in group 2 than in group 1 at eight weeks (p < 0.012). The ultimate load to failure increased with the number of suture anchors used immediately post-operatively, and at four and eight weeks. The increased load to failure at eight weeks seemed to be related to the increase in the surface area of healed tendon-to-bone in the double-row repair group


Bone & Joint Research
Vol. 6, Issue 2 | Pages 90 - 97
1 Feb 2017
Rajfer RA Kilic A Neviaser AS Schulte LM Hlaing SM Landeros J Ferrini MG Ebramzadeh E Park S

Objectives. We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Materials and Methods. Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. Results. When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength (t-test, p = 0.029) and 92% higher stiffness (t-test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. Conclusion. This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression. Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:–97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 11 | Pages 1545 - 1550
1 Nov 2007
Koslowsky TC Mader K Dargel J Koebke J Hellmich M Pennig D

We have evaluated four different fixation techniques for the reconstruction of a standard Mason type-III fracture of the radial head in a sawbone model. The outcome measurements were the quality of the reduction, and stability. A total of 96 fractures was created. Six surgeons were involved in the study and each reconstructed 16 fractures with 1.6 mm fine-threaded wires (Fragment Fixation System (FFS)), T-miniplates, 2 mm miniscrews and 2 mm Kirschner (K-) wires; four fractures being allocated to each method using a standard reconstruction procedure. The quality of the reduction was measured after definitive fixation. Biomechanical testing was performed using a transverse plane shear load in two directions to the implants (parallel and perpendicular) with respect to ultimate failure load and displacement at 50 N. A significantly better quality of reduction was achieved using the FFS wires (Tukey’s post hoc tests, p < 0.001) than with the other devices with a mean step in the articular surface and the radial neck of 1.04 mm (. sd. 0.96) for the FFS, 4.25 mm (. sd. 1.29) for the miniplates, 2.21 mm (. sd. 1.06) for the miniscrews and 2.54 mm (. sd. 0.98) for the K-wires. The quality of reduction was similar for K-wires and miniscrews, but poor for miniplates. The ultimate failure load was similar for the FFS wires (parallel, 196.8 N (. sd. 46.8), perpendicular, 212.5 N (. sd. 25.6)), miniscrews (parallel, 211.8 N (. sd. 47.9), perpendicular, 208.0 N (. sd. 65.9)) and K-wires (parallel, 200.4 N (. sd. 54.5), perpendicular, 165.2 N (. sd. 37.9)), but significantly worse (Tukey’s post hoc tests, p < 0.001) for the miniplates (parallel, 101.6 N (. sd. 43.1), perpendicular, 122.7 N (. sd. 40.7)). There was a significant difference in the displacement at 50 N for the miniplate (parallel, 4.8 mm (. sd. 2.8), perpendicular, 4.8 mm (. sd. 1.7)) vs FFS (parallel, 2.1 mm (. sd. 0.8), perpendicular, 1.9 mm (. sd. 0.7)), miniscrews (parallel, 1.8 mm (. sd. 0.5), perpendicular, 2.3 mm (. sd. 0.8)) and K-wires (parallel, 2.2 mm (. sd. 1.8), perpendicular, 2.4 mm (. sd. 0.7; Tukey’s post hoc tests, p < 0.001)). The fixation of a standard Mason type-III fracture in a sawbone model using the FFS system provides a better quality of reduction than that when using conventional techniques. There was a significantly better stability using FFS implants, miniscrews and K-wires than when using miniplates


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 11 | Pages 1614 - 1620
1 Nov 2010
Fini M Tschon M Ronchetti M Cavani F Bianchi G Mercuri M Alberghini M Cadossi R

Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells. Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing. After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times. Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases


The Journal of Bone & Joint Surgery British Volume
Vol. 84-B, Issue 4 | Pages 571 - 578
1 May 2002
Ochi M Uchio Y Kawasaki K Wakitani S Iwasa J

We investigated the clinical, arthroscopic and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 knees (26 patients) over a minimum period of 25 months. Transplantation eliminated locking of the knee and reduced pain and swelling in all patients. The mean Lysholm score improved significantly. Arthroscopic assessment indicated that 26 knees (93%) had a good or excellent outcome. There were few adverse features, except for marked hypertrophy of the graft in three knees, partial detachment of the periosteum in three and partial ossification of the graft in one. Biomechanical tests revealed that the transplants had acquired a hardness similar to that of the surrounding cartilage. We conclude that transplanting chondrocytes in a newly-formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee


The Journal of Bone & Joint Surgery British Volume
Vol. 66-B, Issue 5 | Pages 672 - 681
1 Nov 1984
Shino K Kawasaki T Hirose H Gotoh I Inoue M Ono K

The revascularisation and remodelling of allografts used to replace the anterior cruciate ligament in the canine knee were studied by microangiographic, histological and biomechanical methods. The 26 allografts were obtained from the patellar tendons of other dogs and were stored by deep freezing. In a control study a strip of patellar tendon from the same leg was used as an autologous free graft. Microangiography showed that the allografts had been revascularised from the sixth postoperative week, and had later developed an intrinsic vascular pattern similar to that of a normal anterior cruciate ligament. Histologically, the allograft regained a fibrous framework similar to that of a normal ligament, and showed no evidence of immunological rejection. Biomechanical tests on the allograft replacements showed that their mean maximum tensile strength at 30 weeks was about 30% of that of the control ligaments. There were no significant differences between the mechanical properties of the allografts and the autografts


The Journal of Bone & Joint Surgery British Volume
Vol. 88-B, Issue 5 | Pages 692 - 695
1 May 2006
Karataglis D Kapetanos G Lontos A Christodoulou A Christoforides J Pournaras J

The aim of this biomechanical study was to investigate the role of the dorsal vertebral cortex in transpedicular screw fixation. Moss transpedicular screws were introduced into both pedicles of each vertebra in 25 human cadaver vertebrae. The dorsal vertebral cortex and subcortical bone corresponding to the entrance site of the screw were removed on one side and preserved on the other. Biomechanical testing showed that the mean peak pull-out strength for the inserted screws, following removal of the dorsal cortex, was 956.16 N. If the dorsal cortex was preserved, the mean peak pullout strength was 1295.64 N. The mean increase was 339.48 N (26.13%; p = 0.033). The bone mineral density correlated positively with peak pull-out strength. Preservation of the dorsal vertebral cortex at the site of insertion of the screw offers a significant increase in peak pull-out strength. This may result from engagement by the final screw threads in the denser bone of the dorsal cortex and the underlying subcortical area. Every effort should be made to preserve the dorsal vertebral cortex during insertion of transpedicular screws


Bone & Joint 360
Vol. 12, Issue 6 | Pages 31 - 34
1 Dec 2023

The December 2023 Shoulder & Elbow Roundup360 looks at: Clavicle fractures: is the evidence changing practice?; Humeral shaft fractures, and another meta-analysis…let’s wait for the trials now!; Hemiarthroplasty or total elbow arthroplasty for distal humeral fractures…what does the registry say?; What to do with a first-time shoulder dislocation?; Deprivation indices and minimal clinically important difference for patient-reported outcomes after arthroscopic rotator cuff repair; Prospective randomized clinical trial of arthroscopic repair versus debridement for partial subscapularis tears; Long-term follow-up following closed reduction and early movement for simple dislocation of the elbow; Sternoclavicular joint reconstruction for traumatic acute and chronic anterior and posterior instability.


Bone & Joint Research
Vol. 12, Issue 8 | Pages 504 - 511
23 Aug 2023
Wang C Liu S Chang C

Aims

This study aimed to establish the optimal fixation methods for calcaneal tuberosity avulsion fractures with different fragment thicknesses in a porcine model.

Methods

A total of 36 porcine calcanea were sawed to create simple avulsion fractures with three different fragment thicknesses (5, 10, and 15 mm). They were randomly fixed with either two suture anchors or one headless screw. Load-to-failure and cyclic loading tension tests were performed for the biomechanical analysis.


Bone & Joint Research
Vol. 12, Issue 3 | Pages 219 - 230
10 Mar 2023
Wang L Li S Xiao H Zhang T Liu Y Hu J Xu D Lu H

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.

Methods

C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs.


The Journal of Bone & Joint Surgery British Volume
Vol. 82-B, Issue 3 | Pages 436 - 444
1 Apr 2000
van Loon CJM de Waal Malefijt MC Buma P Stolk PWT Verdonschot N Tromp AM Huiskes R Barneveld A

The properties of impacted morsellised bone graft (MBG) in revision total knee arthroplasty (TKA) were studied in 12 horses. The left hind metatarsophalangeal joint was replaced by a human TKA. The horses were then randomly divided into graft and control groups. In the graft group, a unicondylar, lateral uncontained defect was created in the third metatarsal bone and reconstructed using autologous MBG before cementing the TKA. In the control group, a cemented TKA was implanted without the bone resection and grafting procedure. After four to eight months, the animals were killed and a biomechanical loading test was performed with a cyclic load equivalent to the horse’s body-weight to study mechanical stability. After removal of the prosthesis, the distal third metatarsal bone was studied radiologically, histologically and by quantitative and micro CT. Biomechanical testing showed that the differences in deformation between the graft and the control condyles were not significant for either elastic or time-dependent deformations. The differences in bone mineral density (BMD) between the graft and the control condyles were not significant. The BMD of the MBG was significantly lower than that in the other regions in the same limb. Micro CT showed a significant difference in the degree of anisotropy between the graft and host bone, even although the structure of the area of the MBG had trabecular orientation in the direction of the axial load. Histological analysis revealed that all the grafts were revascularised and completely incorporated into a new trabecular structure with few or no remnants of graft. Our study provides a basis for the clinical application of this technique with MBG in revision TKA


Bone & Joint Research
Vol. 12, Issue 5 | Pages 339 - 351
23 May 2023
Tan J Liu X Zhou M Wang F Ma L Tang H He G Kang X Bian X Tang K

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.

Methods

A tendon-bone insertion injury healing model was established in 92 C57BL/6 male mice. All mice were divided into control and training groups by random digital table method. The control group mice had full free activity in the cage, and the training group mice started the treadmill training on postoperative day 7. The quality of tendon-bone insertion healing was evaluated by histology, immunohistochemistry, reverse transcription quantitative polymerase chain reaction, Western blotting, micro-CT, micro-MRI, open field tests, and CatWalk gait and biomechanical assessments.