RESUMO
OBJECTIVE: Acetabular chondral lesions are common in patients with femoroacetabular impingement (FAI) syndrome. The aim of this study was (1) to evaluate the proliferation potential of primary human chondrocytes (hC) derived from both acetabular and femoral site and (2) to validate cellular differentiation during three-dimensional (3D) cultivation as a prerequisite for autologous matrix-assisted cartilage regeneration of the hip joint. METHODS: hC were isolated from cartilage samples obtained from N = 6 patients during offset reconstruction. Proteoglycan content was assessed by Safranin-O staining. Proliferation and cell viability were quantified by microscopic cell counting and Trypan Blue exclusion. Messenger ribonucleic acid (mRNA) expression levels of collagen type 1 and 2, aggrecan (ACAN), and interleukin-1ß (IL-1ß) genes were assessed upon monolayer cultivation, after 48 h/4-10°C - transport simulation and after 14 days of 3D hydrogel cultivation. RESULTS: Primary hC from acetabular and femoral damaged sites were viable. No significant intergroup differences were observed concerning cell viability (>95%) after monolayer cultivation and transport simulation. Harvest yields from acetabular and femoral cartilage samples were comparable to that known from knee joints (mean ± standard deviation (SD), 13.4 × 10(6) ± 5 × 10(6) cells per culture vs 20 × 10(6) cells). Redifferentiation was induced during 3D hydrogel cultivation as observed by increased levels of collagen II (1000-fold) and ACAN (10-fold) gene vs monolayer cultivation (P < 0.001). CONCLUSION: hC derived from damaged acetabular and femoral site are qualified for autologous matrix-assisted cartilage transplantation paving the way for cell-based cartilage regeneration in FAI patients.
Assuntos
Impacto Femoroacetabular , Cartilagem Articular , Condrócitos , Quadril , Humanos , Interleucina-1betaRESUMO
OBJECTIVE: In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. DESIGN: 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). RESULTS: The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. CONCLUSIONS: While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Agrecanas/metabolismo , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Hibridização Genômica Comparativa , Feminino , Genes Ligados ao Cromossomo Y , Humanos , Hibridização in Situ Fluorescente , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Adulto JovemRESUMO
OBJECTIVE: To evaluate the clinical outcome of a hydrogel-based autologous chondrocyte implantation (ACI) for large articular cartilage defects in the knee joint. DESIGN: Prospective, multicenter, single-arm, phase III clinical trial. ACI was performed in 100 patients with focal full-thickness cartilage defects ranging from 4 to 12 cm2 in size. The primary outcome measure was the responder rate at 2 years using the Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Two years after ACI treatment, 93% of patients were KOOS responders having improved by ≥10 points compared with their pre-operative level. The primary endpoint of the study was met and demonstrated that the KOOS response rate is markedly greater than 40% with a lower 95% CI (confidence interval) of 86.1, more than twice the pre-specified no-effect level. KOOS improvement (least squares mean) was 42.0 ± 1.8 points (95% CI between 38.4 and 45.7). Mean changes from baseline were significant in the overall KOOS and in all 5 KOOS subscores from Month 3 (first measurement) to Month 24 (inclusive) (P < 0.0001). The mean MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) score after 24 months reached 80.0 points (95% CI: 70.0-90.0 points) and 92.1 points in lesions ≤ 5 cm2. CONCLUSIONS: Overall, hydrogel-based ACI proved to be a valuable treatment option for patients with large cartilage defects in the knee as demonstrated by early, statistically significant, and clinically meaningful improvement up to 2 years follow-up. Parallel to the clinical improvements, MRI analyses suggested increasing maturation, re-organization, and integration of the repair tissue. TRIAL REGISTRATION: NCT03319797; EudraCT No.: 2016-002817-22.
Assuntos
Cartilagem Articular , Condrócitos , Cartilagem Articular/cirurgia , Humanos , Hidrogéis/uso terapêutico , Articulação do Joelho/cirurgia , Estudos Prospectivos , Transplante Autólogo/métodosRESUMO
Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/farmacologia , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Extratos Celulares/farmacologia , Matriz Extracelular/fisiologia , Engenharia Tecidual/métodos , Adulto , Cartilagem Articular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/ultraestrutura , Feminino , Humanos , MasculinoRESUMO
INTRODUCTION: In 75% of all cases of rheumatoid arthritis the wrist is affected and in 12% is the region of initial manifestation of this chronic inflammatory joint disease. To prevent destruction of the wrist through carpal dislocation, radiolunate and radioscapholunate arthrodeses have increased in importance. METHODS: During a 6.5-year period, 28 radiolunate and 4 radioscapholunate arthrodeses were performed in 30 patients. The indication for operation was progressive carpal translation and increasing subluxation of the wrist in which existent radiological damage had not reached more than grade III according to the classification of Larsen and co-workers. RESULTS: The results obtained during the follow-up study (median: 17.3 months) showed in most treated patients after partial arthrodesis of the wrist no or fewer complaints concerning swelling and pain and an acceptable remaining range of motion of the wrist in everyday life. In patients with preoperatively existing ulnar deviation less than 15 degrees progressive carpal collapse and dislocation could be mostly prevented. CONCLUSION: All told radiolunate and radioscapholunate arthrodeses successfully stabilized the wrist in patients suffering from rheumatoid arthritis.
Assuntos
Artrite Reumatoide/cirurgia , Artrodese/métodos , Osso Semilunar/cirurgia , Complicações Pós-Operatórias/diagnóstico por imagem , Rádio (Anatomia)/cirurgia , Osso Escafoide/cirurgia , Ulna/cirurgia , Articulação do Punho/cirurgia , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Feminino , Seguimentos , Humanos , Osso Semilunar/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Destreza Motora/fisiologia , Medição da Dor , Satisfação do Paciente , Radiografia , Rádio (Anatomia)/diagnóstico por imagem , Amplitude de Movimento Articular/fisiologia , Osso Escafoide/diagnóstico por imagem , Ulna/diagnóstico por imagem , Articulação do Punho/diagnóstico por imagemRESUMO
Postoperative irradiation of the operative field is an established method to prevent heterotopic ossification in total hip arthroplasty. In this study two theoretical dose-equivalent regimens of radiation therapy were compared. Allogenic bone matrix was implanted in both thighs of 50 adult male Wistar rats to induce heterotopic ossification. Immediately after operation the implants of 40 animals were irradiated using a single-dose of 7 Gy or 5 fractions of 2 Gy each. Ten rats served as a controlgroup and did not undergo irradiation. Radiation therapy with 5 x 2 Gy led to a highly significantly better suppression of heterotopic ossification than irradiation with 1 x 7 Gy (p < 0.001; paired-t-test). Single-dose irradiation reduced the mean calcium contents to 138.87 +/- 22.84 micrograms Ca2+/mg implanted bone matrix; fractionated irradiation obtained a reduction to 63.35 +/- 21.16 micrograms Ca2+/mg implanted bone matrix. In thigh implants not exposed to irradiation the mean calcium content was 191.50 +/- 11.46 micrograms Ca2+/mg implanted bone matrix. Radiographically better suppression of bone formation could be documented after irradiation with 5 x 2 Gy compared to 1 x 7 Gy and non-irradiated implants. The histological aspect of the explanted specimens showed quantitatively more new bone formation in the non-irradiated controls than in both irradiation groups. In view of experimentally demonstrated better effects, as well as the reduced side effects, fractionated irradiation appears preferable.
Assuntos
Ossificação Heterotópica/prevenção & controle , Osteogênese/efeitos da radiação , Animais , Artroplastia de Quadril/efeitos adversos , Matriz Óssea/química , Matriz Óssea/efeitos da radiação , Transplante Ósseo , Cálcio/análise , Fracionamento da Dose de Radiação , Masculino , Ossificação Heterotópica/diagnóstico por imagem , Ossificação Heterotópica/etiologia , Ossificação Heterotópica/metabolismo , Cuidados Pós-Operatórios , Radiografia , Dosagem Radioterapêutica , Ratos , Ratos Wistar , Coxa da Perna , Transplante HomólogoAssuntos
Epônimos , Neoplasias Cardíacas , Mixoma , Condroma , Humanos , Leiomiossarcoma , Paraganglioma , Transtornos da Pigmentação , SíndromeRESUMO
AIM: The treatment of large bone defects remains a challenge for the orthopaedic surgeon. Regenerative therapies with the use of mesenchymal stem cells (MSC) may provide an alternative to autogenous bone transplantation, callus distraction or the use of allografts. MATERIAL AND METHODS: On the occasion of an expert workshop of the German Society for Orthopaedic and Trauma Surgery, a literature search regarding studies with the use of MSC was performed to evaluate its potential for future clinical studies. Furthermore, the legislative requirements were examined. RESULTS: Various in vitro and animal studies showed the benefit of MSC in bone regeneration. However, there are sparse data from clinical studies. Due to recent legislative changes there are several regulatory demands to meet if clinical studies are performed with MSC. CONCLUSIONS: For further evaluation of the role of MSC in the treatment of bone defects there is a need for clinical trials. The current paper provides some assistance for the successful application for clinical trials with MSC. Planning and performance of these studies may require early consultation with the regulatory authorities and cooperation of research centres in order to obtain authorisation for the evaluation of MSC. Preclinical data have to be obtained according to good laboratory practice with equivalent protocols that will be used in the clinical trials. In the latter the implementation of the guidelines for good clinical practice are mandatory.
Assuntos
Osso e Ossos/cirurgia , Ensaios Clínicos como Assunto/legislação & jurisprudência , Transplante de Células-Tronco Mesenquimais/legislação & jurisprudência , Procedimentos Ortopédicos/legislação & jurisprudência , Animais , Regeneração Óssea/fisiologia , Transplante Ósseo/legislação & jurisprudência , Comportamento Cooperativo , Modelos Animais de Doenças , Educação , Alemanha , Humanos , Comunicação Interdisciplinar , Programas Nacionais de Saúde/legislação & jurisprudência , Osteogênese por Distração/legislação & jurisprudência , Osteogênese por Distração/métodosRESUMO
Full thickness defects of the articular cartilage in the knee joint have lower regenerative properties compared to chondral lesions of the ankle. In order to avoid early osteoarthritis, symptomatic articular cartilage defects in younger patients should undergo biological reconstruction as early as possible. There are different surgical procedures available to achieve a biological resurfacing of the articular joint line. Numerous animal experiments and clinical studies have shown that early biological reconstruction of circumscribed cartilage defects in the knee is superior to a conservative or delayed operative treatment. This effect refers not only to the defect healing but also to the elimination of changes following secondary osteoarthritis. The different surgical procedures can be differentiated concerning the various indications and the final outcome. Additional malalignment, meniscus tears, and/or ligament instabilities should be treated simultaneously together with the cartilage resurfacing. The mid- and long-term results of the different current techniques are promising, but further modifications and improvements are needed.
Assuntos
Artroplastia/métodos , Cartilagem Articular/lesões , Cartilagem Articular/cirurgia , Fraturas de Cartilagem/cirurgia , Traumatismos do Joelho/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Humanos , Guias de Prática Clínica como Assunto , Padrões de Prática MédicaRESUMO
The bad risk for an early onset of osteoarthritis in the knee increases with the size of a cartilage defect. A collateral meniscus- or ligament-tear will enforce this hazard in addition. In order to avoid such a development, relevant full-thickness cartilage defects should be reconstructed biologically and attendant meniscus- or ligament-tears as well as varus- or valgus deformities should be treated. A number of studies, including some prospective-randomized trials, have shown that autologous chondrocyte transplantation (ACT) is the most reliable procedure for a surgical treatment of full-thickness cartilage defects larger than 4 cm (2) in adults. One disadvantage of ACT is the extensive approach to the joint and often a hypertrophy of the repair tissue. To solve these problems, some different biomaterials for a matrix-assisted ACT have been developed. The scaffold we use has a covering membrane upside and a collagen-sponge carrying the chondrocytes. By means of special surgical instruments a minimally invasive implantation is possible, reducing the side-effects of an extensive approach. Animal studies showed the regeneration of a hyaline cartilage using our described system. However, results of current clinical studies with the different scaffolds must be awaited before an universal application of matrix-assisted ACT can be recommended.
Assuntos
Materiais Biocompatíveis , Cartilagem Articular/cirurgia , Condrócitos/transplante , Procedimentos Ortopédicos/métodos , Adulto , Animais , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Regeneração , Transplante AutólogoRESUMO
QUESTION: The accuracy of ultrasound in the detection of histological changes is a point of discussion. METHOD: In an experimental study we examined on 58 explanted human patellar tendons the correlation between histological and sonographic changes. RESULTS: Ultrasonographic measurements of length, width, thickness and circumference of the ligaments correlated highly to the explants' macroscopic evaluated dimensions. The sonographic lesions were distinguished in changes in form and echogenicity. Macroscopic visible changes in the explanted ligaments could be detected ultrasonographically. In histological aspect 2 tendons and in ultrasonographical aspect 16 tendons were found to be normal in the whole course of the tendons. 14 ligaments showed histological but not ultrasonographical abnormalities. 42 ligaments were conspicuous in both methods of examination. In the course of the tendons a very high correlation (up to 92%) between sonographical and histological findings was detected. At the insertion are of the patellar ligament the correlation was low. CONCLUSION: The performed study shows that ultrasonographically detected lesions of the patellar ligament point to histological changes but no conclusions to the kind of histological changes can be taken. However ultrasonographically found abnormalities correlate in a high proportion to histologically detectable changes in the ligaments. Ultrasonography is a good method to evaluate degenerative symptoms in the patellar ligament.
Assuntos
Traumatismos do Joelho/diagnóstico por imagem , Patela/diagnóstico por imagem , Traumatismos dos Tendões , Traumatismos dos Tendões/diagnóstico por imagem , Adulto , Idoso , Feminino , Humanos , Traumatismos do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Patela/patologia , Valores de Referência , Traumatismos dos Tendões/patologia , Tendões/diagnóstico por imagem , Tendões/patologia , UltrassonografiaRESUMO
Periarticular ossification is one of the main problems of total hip replacement. Irradiation with megavolt photons is known to be a well recognized means of prevention of heterotopic ossification. So far only little scientific basis exists about the most favourable way of radiotherapy for prevention of ectopic ossification. In this study two theoretically equivalent doses are compared. Allogeneic bone matrix was implanted into both thighs of 50 adult male Wistar rats for experimental induction of heterotopic ossification. Immediately after operation the thigh implants were irradiated with a single dose of 7 Gy or a total dose of 10 Gy given in 5 fractions of 2 Gy each. In the model of matrix-induced osteogenesis in rats fractionated irradiation by 5 x 2 Gy leaded to a highly-significant (p = 0.001) better suppression of ectopic ossification compared to irradiation by 1 x 7 Gy. Once-only irradiation with 1 x 7 Gy leads to a reduction of the calcium contents by 27.5%, split irradiation by 5 x 2 Gy obtained a reduction by 66.9% compared to the calcium contents of thigh implants not exposed to radiation. In view of experimentally proven better effects, fractionated irradiation has to be preferred to single dose radiation also considering less side effects in split radiation.
Assuntos
Ossificação Heterotópica/radioterapia , Radioterapia de Alta Energia/métodos , Animais , Osso e Ossos/efeitos da radiação , Cálcio/análise , Cálcio/efeitos da radiação , Masculino , Ossificação Heterotópica/prevenção & controle , Dosagem Radioterapêutica , Ratos , Ratos WistarRESUMO
97 patients were treated with cementless custom-made total hip replacement of the Aldinger type for osteoarthritis of the hip. All were prophylactically treated by radiotherapy with 5 x 2 Gy. 10.3% showed heterotopic ossification. 8 patients showed ossification grade I by the Arcq scale and 2 of grade II. Clinical results according to the Merle d'Aubigné score were good or very good in over 90% at one year postoperatively. One patient showed loosening of the femoral stem caused by sinking-in of the custom-made implant. During the revision procedure there were no signs of radiation induced damage to the implant bed. Negative sequelae to the bony implant bed by prophylactic radiotherapy cannot be detected on radiological assessment. Side effects like delayed wound healing or alteration of local immune status with subsequent infection which are commonly discussed, were not seen. Fractionated irradiation is an effective means of prophylaxis of heterotopic ossification.
Assuntos
Prótese de Quadril , Ossificação Heterotópica/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Radioterapia de Alta Energia/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/cirurgia , Período Pós-Operatório , Estudos Prospectivos , Desenho de Prótese , Dosagem Radioterapêutica , Resultado do TratamentoRESUMO
INTRODUCTION: Results of animal experiments have demonstrated that the osseous integration of non-cemented prostheses can, at the very least temporarily, be impaired by the application of non-steroidal antiphlogistic agents (such as diclofenac). It is the objective of this study to examine whether there is a direct influence of diclofenac used in usual clinical dosages (3 times 50 mg daily) on bone cells and their progenitor cells which would explain the observed slow integration of the prostheses. METHODS: To investigate this, cultivated human in vitro osteoblasts and stromal bone marrow cells were incubated with increasing doses of the medications. Our study focused on the effect of diclofenac application on proliferation and functional metabolism in both cell lines. The measurable maximal plasma concentration 2 h after the application of one tablet Voltaren 50 reached 1.6 micrograms/ml. This correlated with diclofenac concentrations between 1 and 10 ml found in our experiments. The detected values were correlated to the control group (0 microgram/ml diclofenac). RESULTS: The drug effect upon osteoblasts was higher than on progenitor cells. The proliferation of in vitro stromal bone marrow cells, compared to untreated cells, was found to be decreased. We observed a decrease to 82% at a diclofenac concentration of 1 microgram/ml, Osteoblasts exhibited a decrease to 97.5% at the same concentration. The DNA synthesis increased to 118% in stromal bone marrow cells, in osteoblasts to 144%. In contrast, we detected a neglectible decrease to 92% in the collagen synthesis of osteoblasts compared to untreated cells. The synthesis of osteocalcin by osteoblasts increased to 119%. The alkaline phosphatase activity was found to be decreased to 88% in stromal bone marrow cells and increased in osteoblasts to 111%. CONCLUSION: Temporary inhibiting effects on osseous integration in non-cemented prosthesis by diclofenac could be caused by a disturbance in the anabolic bone metabolism, exhibited by an increase of osteoblastic osteocalcin expression. Osteocalcin as a known negative regulator of the osteoneogenesis is most likely inhibiting the collagen matrix deposition.
Assuntos
Artroplastia/métodos , Células da Medula Óssea/efeitos dos fármacos , Diclofenaco/farmacologia , Osteoblastos/efeitos dos fármacos , Idoso , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Articulações/efeitos dos fármacos , Articulações/cirurgia , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/fisiologia , Fenótipo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologiaRESUMO
Since 1995 German health maintenance laws require hospitals to document and code all referrals, admissions and discharges using the 4-digit ICD. Operative procedures are documented and coded using the ICPM. Beginning in January 1996, reimbursement for health services requires a diagnosis-related billing and payment for special procedures. The decision for billing is based on documented diagnosis and therapy. This extended request for documentation makes an online access to diagnosis and therapy with a computer-assisted coding system advisable. In 1996 in our hospital each diagnosis and operation was manually documented and coded on a form. Since the beginning of 1997, documentation and coding has been exclusively computer-assisted. On the basis of documented diagnosis and therapy the computer provides the route of reimbursement. Retrospectively we evaluated the number of charged diagnosis-related billings and payments for special procedures from January to April of 1996 and 1997. It became evident that with computer-assisted documentation and coding the number of detected and charged diagnosis-related billings and payments for special procedures was significantly increased in comparison with the previous year.
Assuntos
Orçamentos , Computação Matemática , Traumatismo Múltiplo/economia , Programas Nacionais de Saúde/economia , Procedimentos Ortopédicos/economia , Mecanismo de Reembolso/economia , Coleta de Dados , Documentação/métodos , Alemanha , Humanos , Traumatismo Múltiplo/cirurgia , Garantia da Qualidade dos Cuidados de Saúde/economiaRESUMO
Although growth factors have been demonstrated during bone healing, their presence has not yet been confirmed in callus distraction. Therefore, in 3 patients we searched for cytokines during callus distraction. Bone biopsies were immuno-histochemically stained for TGF-beta1, IGF-I, TGF-beta type II receptor, IGF receptor, and proliferating cell nuclear antigen (PCNA). Histologically we found immature woven bone in the middle of the callus zone and increasing calcification and lamellar bone in the re-modelling zone. Osteoblasts and fibroblast-like cells in the middle zone, and osteoblasts in all zones stained for TGF-beta and its receptor. The number of positive staining cells related to proliferous activity as assessed both by PCNA, and by bone density in radiographs. IGF-I could be detected everywhere. In conclusion, growth factors are present in bone formation and in areas of re-modelling during callotasis. Their relation to proliferous activity and radiographic density supports their involvement in osteogenesis.
Assuntos
Calo Ósseo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Osteíte/cirurgia , Osteogênese por Distração , Fator de Crescimento Transformador beta/metabolismo , Adulto , Feminino , Fraturas do Fêmur/cirurgia , Fêmur , Consolidação da Fratura/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Reoperação , TíbiaRESUMO
The histological and enzymatic effects of single-dose irradiation of 7 Gray (Gy) versus fractionated irradiation of 5 x 2 Gy on the suppression of heterotopic ossification were examined over a period of 60 days in adult male Wistar rats (n = 57). The standardized osteogenesis model system in rats 19, 10, 11, 16, 19] was used for this purpose. The course of developing ossifications was documented quantitatively and qualitatively by means of quantitative computed tomography/osteodensitometry and digital luminescence radiography. Assessment of the activities of the enzymes alkaline and acid phosphatase throughout the experiment as well as characterization of the isoenzyme of alkaline phosphatase (AP) in connection with histological observations displayed a metaplasia of the ingrowing connective tissue into bone-typical cells during osteoinduction. Thus, the increase of AP is the first sign of a functional transformation of mesenchymal stem cells into chondroid bone cells. The increase in the acid phosphatase level with a maximum of activity between the 15th and 30th day (according to the respective treatment group) is highly suggestive of a remodeling process paralleling incipient chondroclast and osteoclast activity. In the animal groups undergoing irradiation, the above-mentioned increase of enzymes occurred after a delay. Furthermore, the maximum values observed were lower than those in the group not undergoing irradiation. Both findings were more manifest in the animal group which underwent 5 x 2 Gy of radiation than in the group which underwent single-dose irradiation of 7 Gy. Radiation suppresses matrix-induced osteogenesis. The histological and enzymatic course of this process was unchanged in the animals which did not undergo irradiation. However, it was quantitatively reduced and accompanied by a retardation of osteogenesis. Both effects were again reduced with fractionated irradiation of 5 x 2 Gy, which is theoretically dose-equivalent to a 1 x 7 Gy application. Histological examinations revealed damage to the migratory, proliferating mesenchymal stem cell population by irradiation doses which had relatively small effects on preosteoblasts, osteoblasts, chondroblasts and other specialized cell forms. Therefore, it may be concluded that the smaller degree of heterotopic ossification in the irradiated groups was due to damage of and a decrease in the number of mesenchymal stem cells at the implant site. Our results stress the necessity of instituting postoperative irradiation therapy as early as possible to prevent heterotopic ossification. In view of experimentally proven better effects, fractionated irradiation has to be preferred to a dose-equivalent single-dose radiation, especially considering the fewer side-effects noted with fractionated irradiation.
Assuntos
Ossificação Heterotópica/radioterapia , Fosfatase Ácida/análise , Fosfatase Alcalina/análise , Animais , Densidade Óssea , Matriz Óssea/transplante , Ensaios Enzimáticos Clínicos , Fracionamento da Dose de Radiação , Seguimentos , Medições Luminescentes , Masculino , Modelos Animais , Ossificação Heterotópica/diagnóstico , Ossificação Heterotópica/patologia , Intensificação de Imagem Radiográfica , Dosagem Radioterapêutica , Ratos , Ratos Wistar , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antiprotozoários/química , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/classificação , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/classificação , Reações Antígeno-Anticorpo , Antígenos de Protozoários/análise , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Isotipos de Imunoglobulinas/análise , Imuno-Histoquímica , Cinética , Camundongos , Camundongos Endogâmicos CBA , Ratos , Especificidade da Espécie , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologiaRESUMO
Irradiation is a well established therapeutical concept to prevent heterotopic ossification after joint replacement. The influence of irradiation on proliferation of mature osteoblasts and their potential osteoprogenitors, matrix formation and mineralization are not well known in this setting. We therefore studied the effect of different doses of ionizing irradiation on the several steps of osteogenesis in vitro, using cells isolated from the juvenile rat. A colony forming test, the MTT-viability assay, a cell count, measurement of the cellular protein content and alkaline phosphatase activity, as well as determination of in vitro mineralisation have been applied to calvarian osteoblasts, fibroblasts and stromal bone marrow cells. Irradiation results in a dose-dependent suppression of clonogenic activity in all mitotically active cells, but metabolic activity and matrix synthesis were not impaired. In dense cultures alkaline phosphatase expression and in vitro mineralisation were not significantly affected by irradiation. Our experimental in vitro data suggest that irradiation inhibits the initial phase of in vivo osteogenesis due to the cytostatic effect. Postoperative irradiation after THR must therefore take place as early as possible. The homoeostasis of normal, orthotopic bone does not seem to be severely affected by local low-dose irradiation.
Assuntos
Osteogênese/efeitos da radiação , Fosfatase Alcalina/metabolismo , Animais , Densidade Óssea/efeitos da radiação , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Técnicas In Vitro , Osteoblastos/efeitos da radiação , Ratos , Células Estromais/efeitos da radiaçãoRESUMO
In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM beta-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3-6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells.