Tissue engineering of the anterior cruciate ligament-sodium dodecyl sulfate-acellularized and revitalized tendons are inferior to native tendons.

The acellularization of tendons using detergents (sodium dodecyl sulfate, Triton-X, tri-nitro-butyl-phosphate) is a new source of scaffolds for tissue engineering in anterior cruciate ligament (ACL) repair. In vitro testing demonstrated that acellular tendon scaffolds are biocompatible and show good biomechanical properties, but in vivo confirmation of these results is not yet available. Therefore, the aim of this study was to see in vivo if an acellular allogenic construct colonized with autologous fibroblasts improves the quality of ACL reconstruction. ACL replacement was performed in 31 New Zealand White rabbits using a standardized model. Fifteen animals received autologous semitendinosus tendon, whereas 16 animals were treated with a tissue-engineered construct. This construct was made by acellularization of allogenic semitendinosus tendons using sodium dodecyl sulfate and subsequent in vitro colonization with autologous fibroblasts. Eight weeks postoperatively, macroscopic, biomechanical (ultimate load to failure, elongation, stiffness; n = 8/9), and histological (n = 5) examinations were performed. Biomechanical testing showed decreasing strength of the constructs at 8 weeks after implantation compared with the direct postsurgical strength. However, tissue-engineered constructs (F = 19.7 +/- 20.3 N) were significantly weaker than autologous tendons (F = 61.2 +/- 31.2 N). Histologically, the autologous tendons showed signs of partial necrosis and tissue remodeling. The tissue-engineered constructs exhibited an inflammatory reaction and showed both repopulated and acellular regions. In conclusion, in vivo results were much more unfavorable than in vitro results had suggested. Further studies have to be performed to test if modifications of the acellularization process yield better results in vivo.

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector.

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.

Heterotopic ossification after minimally invasive rotator cuff repair.

Heterotopic ossification is a common phenomenon after spinal cord injury, head injury, neurologic disorders, burns and other trauma, and joint arthroplasty. Periarticular ossifications after shoulder surgery have been known to occur since the 19th century, at an incidence of up to 27%. After arthroscopic and minimally invasive shoulder surgical procedures were introduced and came into broad use, reports about heterotopic ossification became very rare. We describe here a case of heterotopic bone formation in the subdeltoid fascia after arthroscopic subacromial decompression, acromioclavicular joint resection, and mini-open rotator cuff reconstruction were performed with 2 absorbable suture anchors 3 months postoperatively. Computed tomography (CT) confirmed a massive heterotopic ossification of the deltoid muscle. During revision surgery, a 4 x 6.5-cm bone shell that consisted primarily of immature trabecular bone and lamellar bone in smaller proportions was removed. The case presented here is unique in the scientific literature. Although risk factors have been identified, the underlying pathomorphogenetic mechanism of such heterotopic bone formation remains unclear. Prophylactic administration of nonsteroidal anti-inflammatory drugs (NSAIDs) or radiation for arthroscopic or minimally invasive shoulder surgery is not justified, given the low incidence of heterotopic ossification and the known adverse effects. Apparently, information on basic science and on evidence-based therapy is lacking.

Osteochondral transplantation to treat osteochondral lesions in the elbow.

BACKGROUND: Effective treatment of osteochondral lesions in the elbow remains challenging. Arthroscopic débridement and microfracture or retrograde drilling techniques are often insufficient and provide only temporary symptomatic relief. The purpose of this study was to evaluate the treatment of these lesions with osteochondral autografts. METHODS: From 1999 to 2002, seven patients with osteochondral lesions of the capitellum humeri (five patients), trochlea (one patient), or radial head (one patient) were treated with cylindrical osteochondral grafts, which were harvested from the non-weight-bearing area of the proximal aspect of the lateral femoral condyle. The patients (three female and four male patients with an average age of seventeen years) were evaluated preoperatively and postoperatively, with an average follow-up of fifty-nine months. The Broberg and Morrey score was chosen for functional evaluation of the elbow (with regard to motion, pain, strength, activities of daily living, and stability), and the American Shoulder and Elbow Surgeons score was used for the analysis of pain. All patients had imaging studies done preoperatively to evaluate the defect and postoperatively to assess the ingrowth and viability of the graft. The ipsilateral knee was examined for donor-site morbidity. RESULTS: The Broberg and Morrey score improved from a mean (and standard deviation) of 76.3 +/- 13.2 preoperatively to 97.6 +/- 2.7 postoperatively, and pain scores were significantly reduced (p < 0.05). The mean elbow extension lag of 4.7 degrees +/- 5.8 degrees was reduced to 0 degrees postoperatively. Compared with the contralateral side, there was a mean preoperative flexion lag of 12.9 degrees +/- 13.8 degrees . At the time of the final follow-up, flexion was free and was equal bilaterally in all patients. None of the plain radiographs made at the time of follow-up showed any degenerative changes or signs of osteoarthritis. The postoperative magnetic resonance imaging scans showed graft viability and a congruent chondral surface in all seven patients. No donor-site morbidity was noted at one year postoperatively. CONCLUSIONS: The osteochondral autograft procedure described in the present study provides the opportunity to retain viable hyaline cartilage for the repair of osteochondral lesions in the elbow while restoring joint congruity and function and perhaps reducing the risk of osteoarthritis. These medium-term results suggest that the risks of a two-joint procedure are modest and justifiable. In addition, the described technique provides an option for revision surgery after the failure of other surgical procedures.

In vitro analysis of an allogenic scaffold for tissue-engineered meniscus replacement.

Scaffolds play a key role in the field of tissue engineering. Particularly for meniscus replacement, optimal scaffold properties are critical. The aim of our study was to develop a novel scaffold for replacement of meniscal tissue by means of tissue engineering. Emphasis was put on biomechanical properties comparable to native meniscus, nonimmunogenecity, and the possibility of seeding cells into and cultivating them within the scaffold (nontoxicity). For this purpose, native ovine menisci were treated in vitro in a self-developed enzymatic process. Complete cell removal was achieved and shown both histologically and electron microscopically (n = 15). Immunohistochemical reaction (MHC 1/MHC 2) was positive for native ovine meniscus and negative for the scaffold. Compared to native meniscus (25.8 N/mm) stiffness of the scaffold was significantly increased (30.2 N/mm, p < 0.05, n = 10). We determined the compression (%) of the native meniscus and the scaffold under a load of 7 N. The compression was 23% for native meniscus and 29% for the scaffold (p < 0.05, n = 10). Residual force of the scaffold was significantly lower (5.2 N vs. 4.9 N, p < 0.05, n = 10). Autologous fibrochondrocytes were needle injected and successfully cultivated within the scaffolds over a period of 4 weeks (n = 10). To our knowledge, this study is the first to remove cells and immunogenetic proteins (MHC 1/MHC 2) completely out of native meniscus and preserve important biomechanical properties. Also, injected cells could be successfully cultivated within the scaffold. Further in vitro and in vivo animal studies are necessary to establish optimal cell sources, sterilization, and seeding techniques. Cell differentiation, matrix production, in vivo remodeling of the construct, and possible immunological reactions after implantation are subject of further studies.

In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects.

To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.

Tissue engineering of the anterior cruciate ligament: a new method using acellularized tendon allografts and autologous fibroblasts.

INTRODUCTION: The availability of autogenous tendons (middle part of patellar tendon, semitendinosus/gracilis, or quadriceps tendon) for cruciate ligament reconstructions is restricted and related to withdrawal morbidity. Allografts and synthetic ligament materials often show problems regarding long-term stability and immunological reactions. Therefore, the aim of this study was to develop and characterize a new scaffold based on acellular allografts seeded with autologous cells for tissue engineering of the anterior cruciate ligament (ACL). MATERIALS AND METHODS: Semitendinosus tendons of New Zealand White (NZW) rabbits were harvested and acellularized using the detergent sodium dodecyle sulfate (SDS) as the main ingredient. After that, cultured (37 degrees C, 5% CO(2), medium) dermal fibroblasts were injected into the tendons. These constructs were further cultivated for 4, 7, or 14 days under the same culture conditions. Native, acellular, and seeded tendons underwent biomechanical testing (ultimate load to failure [N], stiffness [N/mm], and elongation [%], each n = 9] and histological hematoxylin-eosin (H.E.) staining. Detailed immunohistochemical (collagen I, III, IV, VI, pro-collagen I, versican, and vimentin) analyses were conducted to detect changes in the composition and structure of the extracellular matrix (ECM) after acellularization. RESULTS: Histologically, a cell-free, crimped slack tendon structure after acellularization and a good integration of the cells after injection (4, 7, and 14 days) were seen. Metabolic activity of the seeded cells was demonstrated by positive immunohistochemical staining for pro-collagen I, which was negative in nonseeded constructs. Major differences in staining patterns of the various other ECM components were not observed. Biomechanically, the maximum load to failure of these tendons was comparable to native tendons (P = 0.429; native 134.5 +/- 12.9 N; acellular 118.5 +/- 7.3 N; seeded 132.3 +/- 5.6 N). Stiffness and elongation were comparable between native and acellular tendons, but differed significantly after seeding (P < 0.001). CONCLUSION: The described method is suitable to make tendons completely cell free without changing their major biomechanical properties. Preservation of the ECM and of the collagen fiber structure by this method should give an ideal environment for autologous cell integration and metabolic activity in contrast to other approaches for tissue acellularization. The cell disruption and extraction of cell detritus should minimize adverse immunogenic reactions.

Arthroscopic reconstruction of an isolated avulsion fracture of the lesser tuberosity.

Isolated avulsion fracture of the lesser tuberosity represents an extremely rare injury that occurs mainly in younger patients. This report outlines the case of a 35-year-old healthy man who sustained a bony avulsion of the subscapularis tendon during an abduction external rotation trauma to his left shoulder. The injury was diagnosed by physical examination, standard radiographs, and magnetic resonance imaging. An arthroscopic reconstruction including closed reduction and internal fixation using suture anchors was performed. After a follow-up of 6 months, the patient's Constant Score increased from 62.4 points preoperatively to 91.3 points. At the latest follow-up, the patient was completely pain free, had regained a full work activity level, and returned to overhead sports. On postoperative standard radiographs and magnetic resonance images, the bony fragment was shown to be consolidated in an anatomic position. In cases of displaced isolated fractures of the lesser tuberosity without significant subluxation or dislocation of the long head of the biceps arthroscopic reconstruction using suture anchors can lead to a good clinical and radiological result.

Meniscus allograft transplantation using posterior peripheral suture technique: a preliminary follow-up study.

The aim of this study was to describe the indication, planning, technique, rehabilitation, and clinical results after cryopreserved allograft meniscus transplantation. Forty consecutive patients, 33 men and 7 women (mean, 37.3 years of age), were evaluated at 1-year follow-up post surgery. Symptoms, patient satisfaction, ROM (range of motion), surgical time, blood loss, and surgical history were evaluated. Thirty-eight (95%) patients had previous total or partial meniscectomy (mean, 11.4 years ago). Preoperatively, chief complaints were knee joint line pain and swelling. Mean surgical time and blood loss were 123 min and 87 g, respectively. At 12 months postsurgery, 5% and 10%, respectively, complained of pain and swelling; ROM was 0 degrees -132 degrees. Thirty-eight (95%) patients were satisfied. According to the results, meniscus transplantation can lead to significant pain relief and satisfaction in young symptomatic meniscectomized patients. However, long-term results must be obtained to prove the effectiveness of this technique in prevention of degenerative joint changes.

Muscle-based gene therapy and tissue engineering for treatment of growth plate injuries.

Growth plate injuries may lead to a progressive angular deformity or longitudinal growth disturbance. The authors investigated the feasibility of gene therapy and tissue engineering based on autologous muscle-and adenoviral-mediated gene transfer of insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) to treat tibial physeal defects in rabbits. The medial half of the left proximal tibial growth plate was completely excised in 44 6-week-old New Zealand white rabbits. Four experimental groups were created: no treatment (I), autologous muscle interposition (II), autologous muscle interposition injected with adIGF-1 (III), and autologous muscle interposition injected with adBMP-2 (IV). Radiographic and histologic assessments were obtained postoperatively. Significant tibial shortening and a compact osseous bridge were observed in groups I and IV. Growth plates remained open in groups II and III. This experiment demonstrates that IGF-1 had a supportive effect on physeal chondrocytes, while BMP-2 caused increased osteogenic activity in the injured growth plates.

Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study.

BACKGROUND: The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits. METHODS: The anterior cruciate ligaments of adult New Zealand White rabbits were replaced with autologous double-bundle semitendinosus tendon grafts. The semitendinosus tendon grafts had been infected in vitro with adenovirus-luciferase, adenovirus-LacZ (AdLacZ), or adenovirus-BMP-2 (AdBMP-2); untreated grafts served as controls. The grafts were examined histologically at two, four, six, and eight weeks after surgery. In additional experiments, the structural properties of the femur-anterior cruciate ligament graft-tibia complexes, from animals killed eight weeks postoperatively, were determined from uniaxial tests. The stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. RESULTS: Genetically engineered semitendinosus tendon grafts expressed reporter genes as well as BMP-2 in vitro. The AdLacZ-infected grafts showed two different histological patterns of transduction. Intra-articularly, infected cells were mostly aligned along the surface, and they decreased in number between two and eight weeks after surgery. In the intra-tunnel portions of the grafts, the number of infected cells did not decrease during the observation period. Moreover, a high number of transduced cells was found in the deeper layers of the tendons. In the control group, granulation-type tissue at the tendon-bone interface showed progressive reorganization into a dense connective tissue, and a later establishment of fibers resembling Sharpey fibers. In the specimens with an AdBMP-2-infected anterior cruciate ligament graft, a broad zone of newly formed matrix resembling chondro-osteoid had formed at the tendon-bone interface at four weeks after surgery. This area was increased at six weeks, showing a transition from bone to mineralized cartilage and nonmineralized fibrocartilage. In addition, in the AdBMP-2-treated specimens, the tendon-bone interface in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness (29.0 +/- 7.1 N/mm compared with 16.7 +/- 8.3 N/mm) and the ultimate load to failure (108.8 +/- 50.8 N compared with 45.0 +/- 18.0 N) were significantly enhanced in the specimens with an AdBMP-2-transduced graft when compared with the control values (p < 0.05). CONCLUSION: This study demonstrates that BMP-2 gene transfer significantly improves the integration of semitendinosus tendon grafts in bone tunnels after reconstruction of the anterior cruciate ligament in rabbits. CLINICAL RELEVANCE: Novel technologies including gene therapy and tissue engineering, such as those described in this study, may provide useful therapeutic procedures to enhance biological healing after reconstruction of the anterior cruciate ligament.

Genetic engineering of meniscal allografts.

Allograft meniscal transplantation represents one of the few available treatment options after menisectomy. Despite acceptable early results, a considerable controversy exists with regard to poor graft regeneration, shrinkage and biomechanical failure of transplanted menisci. Transfer of specific growth factor genes may improve the regeneration process of meniscal allografts. The aim of this study was to investigate the feasibility of gene transfer in meniscal allografts in rabbits. Four different viral vectors encoding marker genes, including lacZ, luciferase, and green fluorescence protein were used to investigate viral transduction in 50 lapine menisci for 4 weeks in vitro. Subsequently, 16 unilateral meniscus replacements were performed with ex vivo retrovirally transduced meniscal allografts, and the expression of the lacZ gene was examined histologically at 2, 4, 6, and 8 weeks after transplantation. Gene expression in the superficial cell layers of the menisci can be detected for up to 4 weeks in vitro, but the level of gene transfer declined over time. The transduction with retrovirus showed better persistence and deep penetration of the menisci with infected cells. In vivo, declining numbers of beta-galactosidase-positive cells were also detected in retrovirally transduced allografts up to 8 weeks. Consistently, transduced cells were found at the menisco-synovial junction of the transplants and in deeper layers of the menisci. There was no evidence of cellular immune response in the transduced transplants. This investigation showed a prospective for growth factor delivery in auto- and allografts. In further experiments, vectors expressing therapeutic proteins such as growth factors will be investigated to assess their potential to improve remodeling and healing of meniscal allografts.

Nuevas lineas de terapia genetica en medicina del deporte / Gene therapy in sport medicine

En las ultimas 2 decadas, el tratamiento de las lesiones deportivas ha mejorado a traves de adelantos en los programas de rehabilitacion y de las tecnicas quirurgicas. A pesar de estos avances, no se encontro un tratamiento optimo para las lesiones de los ligamentos cruzados, para las lesiones centrales de meniscos, las lesiones del cartilago articular o retardos de consolidacion de fracturas debido a la pobre capacidad de curacion de estos tejidos. Nuevos acercamientos biologicos se focalizan en el tratamiento de dichas lesiones utilizando factores de crecimiento celular que estimulan el proceso de curacion. Uno de estos promisorios tratamientos a futuro son los relativos a la terapia genetica que intenta transferir genes especificos que incluyan factores de crecimiento proteicos dentro de las celulas del tejido lesionado

Nuevas lineas de terapia genetica en medicina del deporte / Gene therapy in sport medicine

En las ultimas 2 decadas, el tratamiento de las lesiones deportivas ha mejorado a traves de adelantos en los programas de rehabilitacion y de las tecnicas quirurgicas. A pesar de estos avances, no se encontro un tratamiento optimo para las lesiones de los ligamentos cruzados, para las lesiones centrales de meniscos, las lesiones del cartilago articular o retardos de consolidacion de fracturas debido a la pobre capacidad de curacion de estos tejidos. Nuevos acercamientos biologicos se focalizan en el tratamiento de dichas lesiones utilizando factores de crecimiento celular que estimulan el proceso de curacion. Uno de estos promisorios tratamientos a futuro son los relativos a la terapia genetica que intenta transferir genes especificos que incluyan factores de crecimiento proteicos dentro de las celulas del tejido lesionado