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Bone & Joint 360
Vol. 13, Issue 5 | Pages 51 - 52
1 Oct 2024
Marson BA

The Cochrane Collaboration has produced three new reviews relevant to bone and joint surgery since the publication of the last Cochrane Corner. These are relevant to a wide range of musculoskeletal specialists, and include reviews in lateral elbow pain, osteoarthritis of the big toe joint, and cervical spine injury in paediatric trauma patients.


Bone & Joint 360
Vol. 13, Issue 3 | Pages 48 - 49
3 Jun 2024
Marson BA

The Cochrane Collaboration has produced five new reviews relevant to bone and joint surgery since the publication of the last Cochrane Corner These reviews are relevant to a wide range of musculoskeletal specialists, and include reviews in Morton’s neuroma, scoliosis, vertebral fractures, carpal tunnel syndrome, and lower limb arthroplasty.


Bone & Joint 360
Vol. 13, Issue 1 | Pages 44 - 45
1 Feb 2024
Marson BA

This edition of the Cochrane Corner looks at the three reviews that were published in the second half of 2023: surgical versus non-surgical interventions for displaced intra-articular calcaneal fractures; cryotherapy following total knee arthroplasty; and physical activity and education about physical activity for chronic musculoskeletal pain in children and adolescents.


The Journal of Bone & Joint Surgery British Volume
Vol. 80-B, Issue 2 | Pages 365 - 369
1 Mar 1998
Abraham P Leftheriotis G Saumet JL

Chronic compartment syndrome (CCS) is usually considered to be due to ischaemia of muscle. We have attempted to use the direct measurement of muscle blood flow for diagnosis since the assessment of intracompartmental pressure does not provide accurate knowledge of the vascular state. We recorded simultaneously continuous measurements of the laser Doppler flow (LDF) in muscle and the intracompartment pressure (ICP) after exercise in seven patients with CCS, and in seven control subjects. The mean ICP was 74.1 ± 4.4 mmHg in CCS patients and 24.2 ± 3.4 mmHg in control subjects one minute after exercise, decreasing to 34.6 ± 2.3 mmHg and 15.0 ± 1.6 mmHg at 20 min, respectively. The LDF was 0.80 ± 0.11 arbitrary units (AU) in control subjects and 1.09 ± 0.14 AU in CCS patients one minute after exercise, and 0.41 ± 0.11 AU and 0.27 ± 0.04 AU, respectively, at the end of the recovery period. The ICP showed a progressive decrease over time in both groups. The LDF decreased sharply during the first minutes of recovery in control subjects, but in patients with CCS there was a delayed hyperaemic peak with blood flow reaching 0.84 ± 0.10 AU at nine minutes as against 0.33 ± 0 .06 AU for control subjects (p < 0.01). The ICP increased in both control subjects and CCS patients after exercise with no clear cut-off point between the groups. By contrast, changes in muscle blood flow over time were clearly different between control subjects and patients with CCS. For this reason, LDF should be investigated further as a technique for the diagnosis of CCS


Bone & Joint 360
Vol. 13, Issue 4 | Pages 43 - 45
2 Aug 2024
Evans JT Evans JP Whitehouse MR


Bone & Joint Research
Vol. 5, Issue 2 | Pages 61 - 65
1 Feb 2016
Scott EEF Hamilton DF Wallace RJ Muir AY Simpson AHRW

Objectives. Temperature is known to influence muscle physiology, with the velocity of shortening, relaxation and propagation all increasing with temperature. Scant data are available, however, regarding thermal influences on energy required to induce muscle damage. Methods. Gastrocnemius and soleus muscles were harvested from 36 male rat limbs and exposed to increasing impact energy in a mechanical test rig. Muscle temperature was varied in 5°C increments, from 17°C to 42°C (to encompass the in vivo range). The energy causing non-recoverable deformation was recorded for each temperature. A measure of tissue elasticity was determined via accelerometer data, smoothed by low-pass fifth order Butterworth filter (10 kHz). Data were analysed using one-way analysis of variance (ANOVA) and significance was accepted at p = 0.05. Results. The energy required to induce muscle failure was significantly lower at muscle temperatures of 17°C to 32°C compared with muscle at core temperature, i.e., 37°C (p < 0.01). During low-energy impacts there were no differences in muscle elasticity between cold and warm muscles (p = 0.18). Differences in elasticity were, however, seen at higher impact energies (p < 0.02). Conclusion. Our findings are of particular clinical relevance, as when muscle temperature drops below 32°C, less energy is required to cause muscle tears. Muscle temperatures of 32°C are reported in ambient conditions, suggesting that it would be beneficial, particularly in colder environments, to ensure that peripheral muscle temperature is raised close to core levels prior to high-velocity exercise. Thus, this work stresses the importance of not only ensuring that the muscle groups are well stretched, but also that all muscle groups are warmed to core temperature in pre-exercise routines. Cite this article: Professor A. H. R. W. Simpson. Increased risk of muscle tears below physiological temperature ranges. Bone Joint Res 2016;5:61–65. doi: 10.1302/2046-3758.52.2000484


The Journal of Bone & Joint Surgery British Volume
Vol. 81-B, Issue 4 | Pages 700 - 704
1 Jul 1999
Sochart DH Hardinge K

We have studied the relationship between movements of the foot and ankle and venous blood flow from the lower limb using colourflow Duplex ultrasound to determine the optimum type of exercise for promoting venous return. Studies of both active and passive movements were carried out on 40 limbs in 20 subjects (18 men; 2 women), with a median age of 27 years (20 to 54). We assessed ankle dorsiflexion and plantar flexion, subtalar inversion and eversion, and a combination of all movements. There was no difference in venous flow when comparing opposite limbs in a single subject (p > 0.5), but active exercises produced higher peak and mean velocities of blood flow than passive ones. The active combined movement produced the highest velocities with an increase of 38% in mean and of 58% in peak flow velocities, which were significantly greater than the peak and mean flow rates produced by passive movements. The active combined exercise would therefore be the most effective in eliminating stasis and could contribute to the prevention of deep-vein thrombosis


Bone & Joint 360
Vol. 11, Issue 1 | Pages 50 - 51
1 Feb 2022
Das A


Bone & Joint 360
Vol. 10, Issue 4 | Pages 49 - 51
1 Aug 2021
Evans JT Welch M Whitehouse MR


Bone & Joint 360
Vol. 10, Issue 3 | Pages 38 - 39
1 Jun 2021
Das A


The Journal of Bone & Joint Surgery British Volume
Vol. 82-B, Issue 8 | Pages 1201 - 1203
1 Nov 2000
Karrholm J Brandsson S Freeman MAR

We studied the knees of 11 volunteers using RSA during a step-up exercise requiring extension while weight-bearing from 50° to 0°. The findings on weight-bearing flexion with and without external rotation of the tibia based on MRI were confirmed


The Journal of Bone & Joint Surgery British Volume
Vol. 82-B, Issue 4 | Pages 591 - 594
1 May 2000
Milgrom C Finestone A Simkin A Ekenman I Mendelson S Millgram M Nyska M Larsson E Burr D

Mechanical loading during physical activity produces strains within bones. It is thought that these forces provide the stimulus for the adaptation of bone. Tibial strains and rates of strain were measured in vivo in six subjects during running, stationary bicycling, leg presses and stepping and were compared with those of walking, an activity which has been found to have only a minimal effect on bone mass. Running had a statistically significant higher principal tension, compression and shear strain and strain rates than walking. Stationary bicycling had significantly lower tension and shear strains than walking. If bone strains and/or strain rates higher than walking are needed for tibial bone strengthening, then running is an effective strengthening exercise for tibial bone


Bone & Joint 360
Vol. 9, Issue 3 | Pages 44 - 45
1 Jun 2020
Das MA


Bone & Joint 360
Vol. 9, Issue 1 | Pages 51 - 52
1 Feb 2020
Das A


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


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


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


Bone & Joint 360
Vol. 7, Issue 2 | Pages 40 - 42
1 Apr 2018
Foy MA


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 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 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.


Bone & Joint Research
Vol. 1, Issue 11 | Pages 297 - 309
1 Nov 2012
McIlwraith CW Frisbie DD Kawcak CE

Osteoarthritis (OA) is an important cause of pain, disability and economic loss in humans, and is similarly important in the horse. Recent knowledge on post-traumatic OA has suggested opportunities for early intervention, but it is difficult to identify the appropriate time of these interventions. The horse provides two useful mechanisms to answer these questions: 1) extensive experience with clinical OA in horses; and 2) use of a consistently predictable model of OA that can help study early pathobiological events, define targets for therapeutic intervention and then test these putative therapies. This paper summarises the syndromes of clinical OA in horses including pathogenesis, diagnosis and treatment, and details controlled studies of various treatment options using an equine model of clinical OA.


Bone & Joint 360
Vol. 3, Issue 5 | Pages 36 - 37
1 Oct 2014
Di Martino A


The Bone & Joint Journal
Vol. 96-B, Issue 12 | Pages 1578 - 1585
1 Dec 2014
Rankin KS Sprowson AP McNamara I Akiyama T Buchbinder R Costa ML Rasmussen S Nathan SS Kumta S Rangan A

Trauma and orthopaedics is the largest of the surgical specialties and yet attracts a disproportionately small fraction of available national and international funding for health research. With the burden of musculoskeletal disease increasing, high-quality research is required to improve the evidence base for orthopaedic practice. Using the current research landscape in the United Kingdom as an example, but also addressing the international perspective, we highlight the issues surrounding poor levels of research funding in trauma and orthopaedics and indicate avenues for improving the impact and success of surgical musculoskeletal research.

Cite this article: Bone Joint J 2014; 96-B:1578–85.


The Journal of Bone & Joint Surgery British Volume
Vol. 91-B, Issue 3 | Pages 417 - 424
1 Mar 2009
Millar NL Wei AQ Molloy TJ Bonar F Murrell GAC

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry.

We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008).

These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.


The Journal of Bone & Joint Surgery British Volume
Vol. 87-B, Issue 2 | Pages 261 - 266
1 Feb 2005
Földhazy Z Arndt A Milgrom C Finestone A Ekenman I

Strains applied to bone can stimulate its development and adaptation. High strains and rates of strain are thought to be osteogenic, but the specific dose response relationship is not known. In vivo human strain measurements have been performed in the tibia to try to identify optimal bone strengthening exercises for this bone, but no measurements have been performed in the distal radial metaphysis, the most frequent site of osteoporotic fractures. Using a strain gauged bone staple, in vivo dorsal metaphyseal radial strains and rates of strain were measured in ten female patients during activities of daily living, standard exercises and falls on extended hands. Push-ups and falling resulted in the largest compression strains (median 1345 to 3146 με, equivalent to a 0.1345% to 0.3146% length change) and falling exercises in the largest strain rates (18 582 to 45 954 με/s). On the basis of their high strain and/or strain rates these or variations of these exercises may be appropriate for distal radial metaphyseal bone strengthening.


Bone & Joint 360
Vol. 1, Issue 5 | Pages 34 - 34
1 Oct 2012
Gross AE


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.


The Journal of Bone & Joint Surgery British Volume
Vol. 91-B, Issue 1 | Pages 119 - 123
1 Jan 2009
Benson RT McDonnell SM Rees JL Athanasou NA Carr AJ

We assessed the predictive value of the macroscopic and detailed microscopic appearance of the coracoacromial ligament, subacromial bursa and rotator-cuff tendon in 20 patients undergoing subacromial decompression for impingement in the absence of full-thickness tears of the rotator cuff. Histologically, all specimens had features of degenerative change and oedema in the extracellular matrix. Inflammatory cells were seen, but there was no evidence of chronic inflammation. However, the outcome was not related to cell counts.

At three months the mean Oxford shoulder score had improved from 29.2 (20 to 40) to 39.4 (28 to 48) (p < 0.0001) and at six months to 45.5 (36 to 48) (p < 0.0001). At six months, although all patients had improved, the seven patients with a hooked acromion had done so to a less extent than those with a flat or curved acromion judged by their mean Oxford shoulder scores of 43.5 and 46.5 respectively (p = 0.046). All five patients with partial-thickness tears were within this group and demonstrated less improvement than the patients with no tear (mean Oxford shoulder scores 43.2 and 46.4, respectively, p = 0.04). These findings imply that in the presence of a partial-thickness tear subacromial decompression may require additional specific treatment to the rotator cuff if the outcome is to be improved further.


The Journal of Bone & Joint Surgery British Volume
Vol. 93-B, Issue 1 | Pages 131 - 139
1 Jan 2011
Daugaard H Elmengaard B Andreassen TT Baas J Bechtold JE Soballe K

Impaction allograft is an established method of securing initial stability of an implant in arthroplasty. Subsequent bone integration can be prolonged, and the volume of allograft may not be maintained. Intermittent administration of parathyroid hormone has an anabolic effect on bone and may therefore improve integration of an implant.

Using a canine implant model we tested the hypothesis that administration of parathyroid hormone may improve osseointegration of implants surrounded by bone graft. In 20 dogs a cylindrical porous-coated titanium alloy implant was inserted into normal cancellous bone in the proximal humerus and surrounded by a circumferential gap of 2.5 mm. Morsellised allograft was impacted around the implant. Half of the animals were given daily injections of human parathyroid hormone (1–34) 5 μg/kg for four weeks and half received control injections. The two groups were compared by mechanical testing and histomorphometry. We observed a significant increase in new bone formation within the bone graft in the parathyroid hormone group. There were no significant differences in the volume of allograft, bone-implant contact or in the mechanical parameters.

These findings suggest that parathyroid hormone improves new bone formation in impacted morsellised allograft around an implant and retains the graft volume without significant resorption. Fixation of the implant was neither improved nor compromised at the final follow-up of four weeks.


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 5 | Pages 672 - 685
1 May 2007
Goodrich LR Hidaka C Robbins PD Evans CH Nixon AJ

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model.

A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 107 AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 107 naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated.

Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months.

Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model.

The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 4 | Pages 586 - 594
1 Apr 2010
Sonnabend DH Howlett CR Young AA

The establishment of a suitable animal model of repair of the rotator cuff is difficult since the presence of a true rotator cuff anatomically appears to be restricted almost exclusively to advanced primates. Our observational study describes the healing process after repair of the cuff in a primate model. Lesions were prepared and repaired in eight ‘middle-aged’ baboons. Two each were killed at four, eight, 12 and 15 weeks post-operatively. The bone-tendon repair zones were assessed macroscopically and histologically.

Healing of the baboon supraspinatus involved a sequence of stages resulting in the reestablishment of the bone-tendon junction. It was not uniform and occurred more rapidly at the sites of suture fixation than between them. Four weeks after repair the bone-tendon healing was immature. Whereas macroscopically the repair appeared to be healed at eight weeks, the Sharpey fibres holding the repair together did not appear in any considerable number before 12 weeks. By 15 weeks, the bone-tendon junction was almost, but not quite mature.

Our results support the use of a post-operative rehabilitation programme in man which protects the surgical repair for at least 12 to 15 weeks in order to allow maturation of tendon-to-bone healing.


The Journal of Bone & Joint Surgery British Volume
Vol. 90-B, Issue 4 | Pages 527 - 534
1 Apr 2008
Merican AM Amis AA

Anatomical descriptions of the lateral retinaculum have been published, but the attachments, name or even existence of its tissue bands and layers are ill-defined. We have examined 35 specimens of the knee. The deep fascia is the most superficial layer and the joint capsule is the deepest. The intermediate layer is the most substantial and consists of derivatives of the iliotibial band and the quadriceps aponeurosis. The longitudinal fibres of the iliotibial band merge with those of the quadriceps aponeurosis adjacent to the patella. These longitudinal fibres are reinforced by superficial arciform fibres and on the deep aspect by transverse fibres of the iliotibial band. The latter are dense and provide attachment of the iliotibial band to the patella and the tendon of vastus lateralis obliquus.

Our study identifies two important new findings which are a constant connection of the deep fascia to the quadriceps tendon superior and lateral to the patella, and, a connection of the deeper transverse fibres to the tendon of vastus lateralis obliquus.


The Journal of Bone & Joint Surgery British Volume
Vol. 88-B, Issue 9 | Pages 1236 - 1244
1 Sep 2006
Nishimori M Deie M Kanaya A Exham H Adachi N Ochi M

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.