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Bone & Joint Research
Vol. 6, Issue 4 | Pages 231 - 244
1 Apr 2017
Zhang J Yuan T Zheng N Zhou Y Hogan MV Wang JH

Objectives. After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Methods. Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Results. Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Conclusions. Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses. Bone Joint Res 2017;6:231–244. DOI: 10.1302/2046-3758.64.BJR-2017-0268.R1


The Journal of Bone & Joint Surgery British Volume
Vol. 91-B, Issue 8 | Pages 1106 - 1109
1 Aug 2009
Branstetter JG Jackson SR Haggard WO Richelsoph KC Wenke JC

We used a goat model of a contaminated musculoskeletal defect to determine the effectiveness of rapidly-resorbing calcium-sulphate pellets containing amikacin to reduce the local bacterial count. Our findings showed that this treatment eradicated the bacteria quickly, performed as well as standard polymethylmethacrylate mixed with an antibiotic and had many advantages over the latter. The pellets were prepared before surgery and absorbed completely. They released all of the antibiotic and did not require a subsequent operation for their removal. Our study indicated that locally administered antibiotics reduced bacteria within the wound rapidly. This method of treatment may have an important role in decreasing the rate of infection in contaminated wounds


Bone & Joint Research
Vol. 2, Issue 12 | Pages 276 - 284
1 Dec 2013
Karlakki S Brem M Giannini S Khanduja V Stannard J Martin R

Objectives. The period of post-operative treatment before surgical wounds are completely closed remains a key window, during which one can apply new technologies that can minimise complications. One such technology is the use of negative pressure wound therapy to manage and accelerate healing of the closed incisional wound (incisional NPWT). . Methods. We undertook a literature review of this emerging indication to identify evidence within orthopaedic surgery and other surgical disciplines. Literature that supports our current understanding of the mechanisms of action was also reviewed in detail. . Results. A total of 33 publications were identified, including nine clinical study reports from orthopaedic surgery; four from cardiothoracic surgery and 12 from studies in abdominal, plastic and vascular disciplines. Most papers (26 of 33) had been published within the past three years. Thus far two randomised controlled trials – one in orthopaedic and one in cardiothoracic surgery – show evidence of reduced incidence of wound healing complications after between three and five days of post-operative NPWT of two- and four-fold, respectively. Investigations show that reduction in haematoma and seroma, accelerated wound healing and increased clearance of oedema are significant mechanisms of action. . Conclusions. There is a rapidly emerging literature on the effect of NPWT on the closed incision. Initiated and confirmed first with a randomised controlled trial in orthopaedic trauma surgery, studies in abdominal, plastic and vascular surgery with high rates of complications have been reported recently. The evidence from single-use NPWT devices is accumulating. There are no large randomised studies yet in reconstructive joint replacement. Cite this article: Bone Joint Res 2013;2:276–84


Aims

This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

Methods

In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload.


Bone & Joint 360
Vol. 11, Issue 3 | Pages 46 - 47
1 Jun 2022
Das A


Bone & Joint Research
Vol. 4, Issue 3 | Pages 38 - 44
1 Mar 2015
Thornton GM Reno CR Achari Y Morck DW Hart DA

Objectives. Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model. Methods. Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress. Results. Surgical menopause in normal medial collateral ligaments initiated molecular changes in all the categories evaluated. In early healing medial collateral ligaments, surgical menopause resulted in downregulation of specific collagens, proteinases and inflammatory mediators at 6 weeks of healing, and proteoglycans, growth factors and hormone receptors at 14 weeks of healing. Surgical menopause did not produce mechanical changes in normal or early healing medial collateral ligaments. With or without surgical menopause, healing ligaments exhibited increased total creep strain and decreased failure stress compared with uninjured ligaments. Conclusions. Surgical menopause did not affect the mechanical properties of normal or early healing medial collateral ligaments in a rabbit model. The results in this preclinical model suggest that menopause may result in no further impairment to the ligament healing process. . Cite this article: Bone Joint Res 2015;4:38–44


Bone & Joint Research
Vol. 5, Issue 4 | Pages 137 - 144
1 Apr 2016
Paterson SI Eltawil NM Simpson AHRW Amin AK Hall AC

Objectives. During open orthopaedic surgery, joints may be exposed to air, potentially leading to cartilage drying and chondrocyte death, however, the long-term effects of joint drying in vivo are poorly understood. We used an animal model to investigate the subsequent effects of joint drying on cartilage and chondrocytes. Methods. The patellar groove of anaesthetised rats was exposed (sham-operated), or exposed and then subjected to laminar airflow (0.25m/s; 60 minutes) before wounds were sutured and animals recovered. Animals were monitored for up to eight weeks and then sacrificed. Cartilage and chondrocyte properties were studied by histology and confocal microscopy, respectively. Results. Joint drying caused extensive chondrocyte death within the superficial regions of cartilage. Histology of dried cartilage demonstrated a loss of surface integrity at four weeks, fibrillations at eight weeks, and an increased modified Mankin score (p < 0.001). Cartilage thickness increased (p < 0.001), whereas chondrocyte density decreased at four weeks (p < 0.001), but then increased towards sham-operated levels (p < 0.01) at eight weeks. By week eight, chondrocyte pairing/clustering and cell volume increased (p < 0.05; p < 0.001, respectively). Conclusions. These in vivo results demonstrated for the first time that as a result of laminar airflow, cartilage degeneration occurred which has characteristics similar to those seen in early osteoarthritis. Maintenance of adequate cartilage hydration during open orthopaedic surgery is therefore of paramount importance. Cite this article: Dr A. Hall. Drying of open animal joints in vivo subsequently causes cartilage degeneration. Bone Joint Res 2016;5:137–144. DOI: 10.1302/2046-3758.54.2000594


The Bone & Joint Journal
Vol. 96-B, Issue 6 | Pages 845 - 850
1 Jun 2014
Romanò CL Logoluso N Meani E Romanò D De Vecchi E Vassena C Drago L

The treatment of chronic osteomyelitis often includes surgical debridement and filling the resultant void with antibiotic-loaded polymethylmethacrylate cement, bone grafts or bone substitutes. Recently, the use of bioactive glass to treat bone defects in infections has been reported in a limited series of patients. However, no direct comparison between this biomaterial and antibiotic-loaded bone substitute has been performed. . In this retrospective study, we compared the safety and efficacy of surgical debridement and local application of the bioactive glass S53P4 in a series of 27 patients affected by chronic osteomyelitis of the long bones (Group A) with two other series, treated respectively with an antibiotic-loaded hydroxyapatite and calcium sulphate compound (Group B; n = 27) or a mixture of tricalcium phosphate and an antibiotic-loaded demineralised bone matrix (Group C; n = 22). Systemic antibiotics were also used in all groups. After comparable periods of follow-up, the control of infection was similar in the three groups. In particular, 25 out of 27 (92.6%) patients of Group A, 24 out of 27 (88.9%) in Group B and 19 out of 22 (86.3%) in Group C showed no infection recurrence at means of 21.8 (12 to 36), 22.1 (12 to 36) and 21.5 (12 to 36) months follow-up, respectively, while Group A showed a reduced wound complication rate. Our results show that patients treated with a bioactive glass without local antibiotics achieved similar eradication of infection and less drainage than those treated with two different antibiotic-loaded calcium-based bone substitutes. Cite this article: Bone Joint J 2014; 96-B:845–50


Bone & Joint 360
Vol. 9, Issue 5 | Pages 49 - 50
1 Oct 2020
Das MA


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 1 | Pages 159 - 163
1 Jan 2010
Aykut S Öztürk A Özkan Y Yanik K İlman AA Özdemir RM

We studied the effects of coating titanium implants with teicoplanin and clindamycin in 30 New Zealand White rabbits which were randomly assigned to three groups. The intramedullary canal of the left tibia of each rabbit was inoculated with 500 colony forming units of Staphylococcus aureus. Teicoplanin-coated implants were implanted into rabbits in group 1, clindamycin-coated implants into rabbits in group 2, and uncoated implants into those in group 3. All the rabbits were killed one week later. The implants were removed and cultured together with pieces of tibial bone and wound swabs. The rate of colonisation of the organisms in the three groups was compared. Organisms were cultured from no rabbits in group 1, one in group 2 but from all in group 3. There was no significant difference between groups 1 and 2 (p = 1.000). There were significant differences between groups 1 and 3 and groups 2 and 3 (p < 0.001). Significant protection against bacterial colonisation and infection was found with teicoplanin- and clindamycin-coated implants in this experimental model


The Journal of Bone & Joint Surgery British Volume
Vol. 94-B, Issue 6 | Pages 848 - 855
1 Jun 2012
Tayton ER Smith JO Aarvold A Kalra S Dunlop DG Oreffo ROC

When transferring tissue regenerative strategies involving skeletal stem cells to human application, consideration needs to be given to factors that may affect the function of the cells that are transferred. Local anaesthetics are frequently used during surgical procedures, either administered directly into the operative site or infiltrated subcutaneously around the wound. The aim of this study was to investigate the effects of commonly used local anaesthetics on the morphology, function and survival of human adult skeletal stem cells. Cells from three patients who were undergoing elective hip replacement were harvested and incubated for two hours with 1% lidocaine, 0.5% levobupivacaine or 0.5% bupivacaine hydrochloride solutions. Viability was quantified using WST-1 and DNA assays. Viability and morphology were further characterised using CellTracker Green/Ethidium Homodimer-1 immunocytochemistry and function was assessed by an alkaline phosphatase assay. An additional group was cultured for a further seven days to allow potential recovery of the cells after removal of the local anaesthetic. A statistically significant and dose dependent reduction in cell viability and number was observed in the cell cultures exposed to all three local anaesthetics at concentrations of 25% and 50%, and this was maintained even following culture for a further seven days. This study indicates that certain local anaesthetic agents in widespread clinical use are deleterious to skeletal progenitor cells when studied in vitro; this might have relevance in clinical applications


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 2 | Pages 298 - 303
1 Feb 2010
Toom A Suutre S Märtson A Haviko T Selstam G Arend A

We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically. Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (. sd. 4.5) versus 12.7% (. sd. 2.9, p < 0.019), respectively. Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 1 | Pages 116 - 120
1 Jan 2007
Laing AJ Dillon JP Condon E Coffey JC Street JT Wang JH McGuinness AJ Redmond HP

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice. Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1. +. ) and stem cell factor receptor, CD117 (c-kit. +. ) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1. +. mononuclear cell (MNC. sca-1+. ) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC. sca-1+,c-kit+. counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours. Sca-1. +. mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium. Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 11 | Pages 1534 - 1538
1 Nov 2007
Hammer TO Wieling R Green JM Südkamp NP Schneider E Müller CA

This study investigated the quality and quantity of healing of a bone defect following intramedullary reaming undertaken by two fundamentally different systems; conventional, using non-irrigated, multiple passes; or suction/irrigation, using one pass. The result of a measured re-implantation of the product of reaming was examined in one additional group. We used 24 Swiss mountain sheep with a mean tibial medullary canal diameter between 8 mm and 9 mm. An 8 mm ‘napkin ring’ defect was created at the mid-diaphysis. The wound was either surgically closed or occluded. The medullary cavity was then reamed to 11 mm. The Reamer/Irrigator/Aspirator (RIA) System was used for the reaming procedure in groups A (RIA and autofilling) and B (RIA, collected reamings filled up), whereas reaming in group C (Synream and autofilling) was performed with the Synream System. The defect was allowed to auto-fill with reamings in groups A and C, but in group B, the defect was surgically filled with collected reamings. The tibia was then stabilised with a solid locking Unreamed Humerus Nail (UHN), 9.5 mm in diameter. The animals were killed after six weeks. After the implants were removed, measurements were taken to assess the stiffness, strength and callus formation at the site of the defect. There was no significant difference between healing after conventional reaming or suction/irrigation reaming. A significant improvement in the quality of the callus was demonstrated by surgically placing captured reamings into the defect using a graft harvesting system attached to the aspirator device. This was confirmed by biomechanical testing of stiffness and strength. This study suggests it could be beneficial to fill cortical defects with reaming particles in clinical practice, if feasible


Bone & Joint 360
Vol. 7, Issue 6 | Pages 41 - 42
1 Dec 2018
Das A


The Journal of Bone & Joint Surgery British Volume
Vol. 84-B, Issue 5 | Pages 758 - 760
1 Jul 2002
Thornes B Murray P Bouchier-Hayes D

We have compared the rates of infection and resistance in an animal model of an orthopaedic procedure which was contaminated with a low-dose inoculum of Staphylococcus epidermidis. We randomised 44 Sprague-Dawley rats to have bone cement implanted subcutaneously containing either gentamicin or saline (control). The wound was inoculated with a dilute solution of gentamicin-sensitive Staphylococcus epidermidis. At two weeks the cement was retrieved and microbiologically tested. A lower overall rate of infection was seen in the gentamicin-loaded cement group, but there was a significantly higher rate of gentamicin-resistant infection in this group (Fisher’s exact test, p < 0.01). Antibiotic-impregnated cement has an optimum surface for colonisation and prolonged exposure to antibiotic allows mutational resistance to occur. Gentamicin-loaded cement may not be appropriate for revision surgery if it has been used already in previous surgery


The Journal of Bone & Joint Surgery British Volume
Vol. 78-B, Issue 4 | Pages 647 - 651
1 Jul 1996
Arens S Schlegel U Printzen G Ziegler WJ Perren SM Hansis M

Resistance to infection may be influenced by foreign bodies such as devices for fracture fixation. It is known that stainless steel and commercially-pure titanium have different biocompatibilities. We have investigated susceptibility to infection after a local bacterial challenge using standard 2.0 dynamic compression plates of either stainless steel or titanium in rabbit tibiae. After the wounds had been closed, various concentrations of a strain of Staphylococcus aureus were inoculated percutaneously. Under otherwise identical experimental conditions the rate of infection for steel plates (75%) was significantly higher than that for titanium plates (35%) (p < 0.05)


Bone & Joint Research
Vol. 7, Issue 6 | Pages 414 - 421
1 Jun 2018
Yu CD Miao WH Zhang YY Zou MJ Yan XF

Objectives

The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy.

Methods

Human chondrocyte cell line CHON-001 was administrated by different doses of interleukin (IL)-1β to simulate inflammation. Cell viability, migration, apoptosis, IL-6, IL-8, and tumour necrosis factor (TNF)-α expression, as well as expression of apoptosis-related factors, were measured to assess inflammation. miR-126 expression was measured by quantitative polymerase chain reaction (qPCR). Cells were then transfected with miR-126 inhibitor to assess the effect of miR-126 on IL-1β-injured CHON-001 cells. Expression of B-cell lymphoma 2 (Bcl-2) and the activity of mitogen-activated protein kinase (MAPK) / Jun N-terminal kinase (JNK) signaling pathway were measured by Western blot to explore the underlying mechanism through which miR-126 affects IL-1β-induced inflammation.


Bone & Joint 360
Vol. 7, Issue 3 | Pages 38 - 39
1 Jun 2018
Das A


Bone & Joint Research
Vol. 7, Issue 3 | Pages 244 - 251
1 Mar 2018
Tawonsawatruk T Sriwatananukulkit O Himakhun W Hemstapat W

Objectives

In this study, we compared the pain behaviour and osteoarthritis (OA) progression between anterior cruciate ligament transection (ACLT) and osteochondral injury in surgically-induced OA rat models.

Methods

OA was induced in the knee joints of male Wistar rats using transection of the ACL or induction of osteochondral injury. Changes in the percentage of high limb weight distribution (%HLWD) on the operated hind limb were used to determine the pain behaviour in these models. The development of OA was assessed and compared using a histological evaluation based on the Osteoarthritis Research Society International (OARSI) cartilage OA histopathology score.


Bone & Joint Research
Vol. 6, Issue 11 | Pages 631 - 639
1 Nov 2017
Blyth MJG Anthony I Rowe P Banger MS MacLean A Jones B

Objectives

This study reports on a secondary exploratory analysis of the early clinical outcomes of a randomised clinical trial comparing robotic arm-assisted unicompartmental knee arthroplasty (UKA) for medial compartment osteoarthritis of the knee with manual UKA performed using traditional surgical jigs. This follows reporting of the primary outcomes of implant accuracy and gait analysis that showed significant advantages in the robotic arm-assisted group.

Methods

A total of 139 patients were recruited from a single centre. Patients were randomised to receive either a manual UKA implanted with the aid of traditional surgical jigs, or a UKA implanted with the aid of a tactile guided robotic arm-assisted system. Outcome measures included the American Knee Society Score (AKSS), Oxford Knee Score (OKS), Forgotten Joint Score, Hospital Anxiety Depression Scale, University of California at Los Angeles (UCLA) activity scale, Short Form-12, Pain Catastrophising Scale, somatic disease (Primary Care Evaluation of Mental Disorders Score), Pain visual analogue scale, analgesic use, patient satisfaction, complications relating to surgery, 90-day pain diaries and the requirement for revision surgery.


Bone & Joint 360
Vol. 6, Issue 5 | Pages 42 - 44
1 Oct 2017
Ross A


Bone & Joint Research
Vol. 6, Issue 5 | Pages 277 - 283
1 May 2017
Yoshikawa M Nakasa T Ishikawa M Adachi N Ochi M

Objectives

Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine.

Methods

Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs).


Bone & Joint Research
Vol. 6, Issue 3 | Pages 162 - 171
1 Mar 2017
Walker JA Ewald TJ Lewallen E Van Wijnen A Hanssen AD Morrey BF Morrey ME Abdel MP Sanchez-Sotelo J

Objectives

Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis.

Materials and Methods

A total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis.


Bone & Joint Research
Vol. 5, Issue 10 | Pages 500 - 511
1 Oct 2016
Raina DB Gupta A Petersen MM Hettwer W McNally M Tägil M Zheng M Kumar A Lidgren L

Objectives

We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.

Materials and Methods

We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy.


Bone & Joint Research
Vol. 6, Issue 8 | Pages 489 - 498
1 Aug 2017
Mifuji K Ishikawa M Kamei N Tanaka R Arita K Mizuno H Asahara T Adachi N Ochi M

Objectives

The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing.

Methods

Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically.


Bone & Joint Research
Vol. 6, Issue 6 | Pages 358 - 365
1 Jun 2017
Sanghani-Kerai A Coathup M Samazideh S Kalia P Silvio LD Idowu B Blunn G

Objectives

Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration.

Methods

MSCs from young, adult and OVX rats were put in a Boyden chamber to establish their migration towards SDF-1. This was compared with MSCs transfected with CXCR4, as well as MSCs differentiated to osteoblasts.


Bone & Joint Research
Vol. 6, Issue 5 | Pages 315 - 322
1 May 2017
Martinez-Perez M Perez-Jorge C Lozano D Portal-Nuñez S Perez-Tanoira R Conde A Arenas MA Hernandez-Lopez JM de Damborenea JJ Gomez-Barrena E Esbrit P Esteban J

Objectives

Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied.

Methods

We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis.


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.


Bone & Joint Research
Vol. 6, Issue 2 | Pages 73 - 81
1 Feb 2017
Ishihara K Okazaki K Akiyama T Akasaki Y Nakashima Y

Objectives

Osteophytes are products of active endochondral and intramembranous ossification, and therefore could theoretically provide significant efficacy as bone grafts. In this study, we compared the bone mineralisation effectiveness of osteophytes and cancellous bone, including their effects on secretion of growth factors and anabolic effects on osteoblasts.

Methods

Osteophytes and cancellous bone obtained from human patients were transplanted onto the calvaria of severe combined immunodeficient mice, with Calcein administered intra-peritoneally for fluorescent labelling of bone mineralisation. Conditioned media were prepared using osteophytes and cancellous bone, and growth factor concentration and effects of each graft on proliferation, differentiation and migration of osteoblastic cells were assessed using enzyme-linked immunosorbent assays, MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assays, quantitative real-time polymerase chain reaction, and migration assays.


Bone & Joint Research
Vol. 6, Issue 2 | Pages 98 - 107
1 Feb 2017
Kazemi D Shams Asenjan K Dehdilani N Parsa H

Objectives

Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model.

Methods

Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (sd) and compared at different time points between the two groups using the Mann-Whitney U test, with a value < 0.05 considered statistically significant.


Bone & Joint Research
Vol. 5, Issue 12 | Pages 602 - 609
1 Dec 2016
Muto T Kokubu T Mifune Y Inui A Sakata R Harada Y Takase F Kurosaka M

Objectives

Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß.

Methods

Rotator cuff-derived cells were seeded under inflammatory stimulation conditions (with serum-free medium with 1 ng/ml IL-1ß for three hours), and then cultured in different media: serum-free (control group), serum-free + TA (0.1mg/ml) (TA group), serum-free + 10% PRP (PRP group), and serum-free + TA (0.1mg/ml) + 10% PRP (TA+PRP group). Cell morphology, cell viability, and expression of inflammatory and degenerative mediators were assessed.


Bone & Joint Research
Vol. 5, Issue 6 | Pages 218 - 224
1 Jun 2016
Cheng N Guo A Cui Y

Objectives

Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA.

Methods

Collagenase (type II) was injected twice into both knees of three-month-old rabbits to induce OA, combined with two intra–articular injections of Torin 1 (400 nM). Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at eight weeks after injection. Chondrocyte degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13 (MMP-13) and vascular endothelial growth factor (VEGF) expression were analysed by quantitative RT-PCR (qPCR).Beclin-1 and light chain 3 (LC3) expression were examined by Western blotting.


Bone & Joint Research
Vol. 5, Issue 1 | Pages 1 - 10
1 Jan 2016
Burghardt RD Manzotti A Bhave A Paley D Herzenberg JE

Objectives

The purpose of this study was to compare the results and complications of tibial lengthening over an intramedullary nail with treatment using the traditional Ilizarov method.

Methods

In this matched case study, 16 adult patients underwent 19 tibial lengthening over nails (LON) procedures. For the matched case group, 17 patients who underwent 19 Ilizarov tibial lengthenings were retrospectively matched to the LON group.


Bone & Joint Research
Vol. 5, Issue 2 | Pages 33 - 36
1 Feb 2016
Jenkins PJ Morton A Anderson G Van Der Meer RB Rymaszewski LA

Objectives

“Virtual fracture clinics” have been reported as a safe and effective alternative to the traditional fracture clinic. Robust protocols are used to identify cases that do not require further review, with the remainder triaged to the most appropriate subspecialist at the optimum time for review. The objective of this study was to perform a “top-down” analysis of the cost effectiveness of this virtual fracture clinic pathway.

Methods

National Health Service financial returns relating to our institution were examined for the time period 2009 to 2014 which spanned the service redesign.


Bone & Joint Research
Vol. 4, Issue 4 | Pages 56 - 64
1 Apr 2015
Lv YM Yu QS

Objectives

The major problem with repair of an articular cartilage injury is the extensive difference in the structure and function of regenerated, compared with normal cartilage. Our work investigates the feasibility of repairing articular osteochondral defects in the canine knee joint using a composite lamellar scaffold of nano-ß-tricalcium phosphate (ß-TCP)/collagen (col) I and II with bone marrow stromal stem cells (BMSCs) and assesses its biological compatibility.

Methods

The bone–cartilage scaffold was prepared as a laminated composite, using hydroxyapatite nanoparticles (nano-HAP)/collagen I/copolymer of polylactic acid–hydroxyacetic acid as the bony scaffold, and sodium hyaluronate/poly(lactic-co-glycolic acid) as the cartilaginous scaffold. Ten-to 12-month-old hybrid canines were randomly divided into an experimental group and a control group. BMSCs were obtained from the iliac crest of each animal, and only those of the third generation were used in experiments. An articular osteochondral defect was created in the right knee of dogs in both groups. Those in the experimental group were treated by implanting the composites consisting of the lamellar scaffold of ß-TCP/col I/col II/BMSCs. Those in the control group were left untreated.


The Bone & Joint Journal
Vol. 97-B, Issue 2 | Pages 283 - 288
1 Feb 2015
Gupta S Maclean M Anderson JG MacGregor SJ Meek RMD Grant MH

High-intensity narrow-spectrum (HINS) light is a novel violet-blue light inactivation technology which kills bacteria through a photodynamic process, and has been shown to have bactericidal activity against a wide range of species. Specimens from patients with infected hip and knee arthroplasties were collected over a one-year period (1 May 2009 to 30 April 2010). A range of these microbial isolates were tested for sensitivity to HINS-light. During testing, suspensions of the pathogens were exposed to increasing doses of HINS-light (of 123mW/cm2 irradiance). Non-light exposed control samples were also used. The samples were then plated onto agar plates and incubated at 37°C for 24 hours before enumeration. Complete inactivation (greater than 4-log10 reduction) was achieved for all of the isolates. The typical inactivation curve showed a slow initial reaction followed by a rapid period of inactivation. The doses of HINS-light required ranged between 118 and 2214 J/cm2. Gram-positive bacteria were generally found to be more susceptible than Gram-negative.

As HINS-light uses visible wavelengths, it can be safely used in the presence of patients and staff. This unique feature could lead to its possible use in the prevention of infection during surgery and post-operative dressing changes.

Cite this article: Bone Joint J 2015;97-B:283–8.


Bone & Joint Research
Vol. 2, Issue 3 | Pages 58 - 65
1 Mar 2013
Johnson R Jameson SS Sanders RD Sargant NJ Muller SD Meek RMD Reed MR

Objectives

To review the current best surgical practice and detail a multi-disciplinary approach that could further reduce joint replacement infection.

Methods

Review of relevant literature indexed in PubMed.


Bone & Joint Research
Vol. 3, Issue 11 | Pages 310 - 316
1 Nov 2014
Tomaszewski R Bohosiewicz J Gap A Bursig H Wysocka A

Objectives

The aim of this experimental study on New Zealand’s white rabbits was to investigate the transplantation of autogenous growth plate cells in order to treat the injured growth plate. They were assessed in terms of measurements of radiological tibial varus and histological characteristics.

Methods

An experimental model of plate growth medial partial resection of the tibia in 14 New Zealand white rabbits was created. During this surgical procedure the plate growth cells were collected and cultured. While the second surgery was being performed, the autologous cultured growth plate cells were grafted at the right tibia, whereas the left tibia was used as a control group.


Bone & Joint Research
Vol. 3, Issue 2 | Pages 32 - 37
1 Feb 2014
Singh A Goel SC Gupta KK Kumar M Arun GR Patil H Kumaraswamy V Jha S

Introduction

Osteoarthritis (OA) is a progressively debilitating disease that affects mostly cartilage, with associated changes in the bone. The increasing incidence of OA and an ageing population, coupled with insufficient therapeutic choices, has led to focus on the potential of stem cells as a novel strategy for cartilage repair.

Methods

In this study, we used scaffold-free mesenchymal stem cells (MSCs) obtained from bone marrow in an experimental animal model of OA by direct intra-articular injection. MSCs were isolated from 2.8 kg white New Zealand rabbits. There were ten in the study group and ten in the control group. OA was induced by unilateral transection of the anterior cruciate ligament of the knee joint. At 12 weeks post-operatively, a single dose of 1 million cells suspended in 1 ml of medium was delivered to the injured knee by direct intra-articular injection. The control group received 1 ml of medium without cells. The knees were examined at 16 and 20 weeks following surgery. Repair was investigated radiologically, grossly and histologically using haematoxylin and eosin, Safranin-O and toluidine blue staining.


Bone & Joint 360
Vol. 3, Issue 3 | Pages 39 - 40
1 Jun 2014
Arastu M


Bone & Joint Research
Vol. 3, Issue 7 | Pages 236 - 240
1 Jul 2014
Robubi A Berger C Schmid M Huber KR Engel A Krugluger W

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

Methods

Primary human osteoblasts in in vitro culture were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1) or 48 hours (BMP2 and FGF2). Experiments were performed during the exponential growth phase with approximately 1e7 cells per 75 cm2 flask. mRNA was reverse transcribed directly and analysed using RT-PCR Taqman assays. Expression levels of key genes involved in cell growth and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A, LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific Taqman probes.


The Journal of Bone & Joint Surgery British Volume
Vol. 94-B, Issue 4 | Pages 564 - 569
1 Apr 2012
Pendegrass CJ El-Husseiny M Blunn GW

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls.

Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively.


Bone & Joint Research
Vol. 1, Issue 9 | Pages 218 - 224
1 Sep 2012
Tabuchi K Soejima T Kanazawa T Noguchi K Nagata K

Objectives

The purpose of this study was to evaluate chronological changes in the collagen-type composition at tendon–bone interface during tendon–bone healing and to clarify the continuity between Sharpey-like fibres and inner fibres of the tendon.

Methods

Male white rabbits were used to create an extra-articular bone–tendon graft model by grafting the extensor digitorum longus into a bone tunnel. Three rabbits were killed at two, four, eight, 12 and 26 weeks post-operatively. Elastica van Gieson staining was used to colour 5 µm coronal sections, which were examined under optical and polarised light microscopy. Immunostaining for type I, II and III collagen was also performed.


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 5 | Pages 693 - 700
1 May 2007
Ishii I Mizuta H Sei A Hirose J Kudo S Hiraki Y

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant.

Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect.

Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.


Bone & Joint Research
Vol. 1, Issue 6 | Pages 125 - 130
1 Jun 2012
Bøe BG Støen RØ Solberg LB Reinholt FP Ellingsen JE Nordsletten L

Objectives

An experimental rabbit model was used to test the null hypothesis, that there is no difference in new bone formation around uncoated titanium discs compared with coated titanium discs when implanted into the muscles of rabbits.

Methods

A total of three titanium discs with different surface and coating (1, porous coating; 2, porous coating + Bonemaster (Biomet); and 3, porous coating + plasma-sprayed hydroxyapatite) were implanted in 12 female rabbits. Six animals were killed after six weeks and the remaining six were killed after 12 weeks. The implants with surrounding tissues were embedded in methyl methacrylate and grinded sections were stained with Masson-Goldners trichrome and examined by light microscopy of coded sections.


The Journal of Bone & Joint Surgery British Volume
Vol. 94-B, Issue 7 | Pages 998 - 1006
1 Jul 2012
Kodama A Kamei N Kamei G Kongcharoensombat W Ohkawa S Nakabayashi A Ochi M

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 6 | Pages 889 - 893
1 Jun 2010
Kocaoglu B Agir I Nalbantoglu U Karahan M Türkmen M

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 Journal of Bone & Joint Surgery British Volume
Vol. 94-B, Issue 7 | Pages 865 - 874
1 Jul 2012
Mills LA Simpson AHRW

This review is aimed at clinicians appraising preclinical trauma studies and researchers investigating compromised bone healing or novel treatments for fractures. It categorises the clinical scenarios of poor healing of fractures and attempts to match them with the appropriate animal models in the literature.

We performed an extensive literature search of animal models of long bone fracture repair/nonunion and grouped the resulting studies according to the clinical scenario they were attempting to reflect; we then scrutinised them for their reliability and accuracy in reproducing that clinical scenario.

Models for normal fracture repair (primary and secondary), delayed union, nonunion (atrophic and hypertrophic), segmental defects and fractures at risk of impaired healing were identified. Their accuracy in reflecting the clinical scenario ranged greatly and the reliability of reproducing the scenario ranged from 100% to 40%.

It is vital to know the limitations and success of each model when considering its application.


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 6 | Pages 894 - 899
1 Jun 2010
Khattak MJ Ahmad T Rehman R Umer M Hasan SH Ahmed M

The nervous system is known to be involved in inflammation and repair. We aimed to determine the effect of physical activity on the healing of a muscle injury and to examine the pattern of innervation. Using a drop-ball technique, a contusion was produced in the gastrocnemius in 20 rats. In ten the limb was immobilised in a plaster cast and the remaining ten had mobilisation on a running wheel. The muscle and the corresponding dorsal-root ganglia were studied by histological and immunohistochemical methods.

In the mobilisation group, there was a significant reduction in lymphocytes (p = 0.016), macrophages (p = 0.008) and myotubules (p = 0.008) between three and 21 days. The formation of myotubules and the density of nerve fibres was significantly higher (both p = 0.016) compared with those in the immobilisation group at three days, while the density of CGRP-positive fibres was significantly lower (p = 0.016) after 21 days.

Mobilisation after contusional injury to the muscle resulted in early and increased formation of myotubules, early nerve regeneration and progressive reduction in inflammation, suggesting that it promoted a better healing response.