RESUMO
PURPOSE: A histological study of a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. METHODS: Bilateral fresh-frozen cadaveric knees of two male donors (age 71 and 76 years) with no history of prior knee injury were examined. All dissections were performed by one experienced orthopaedic surgeon. Haematoxylin and Eosin staining was used to reveal tissue morphology. Goldner trichrome staining was used to evaluate the connective tissue. S100 and PGP 9.5 labelling were used for immunohistochemical analysis. RESULTS: In all cadaveric knees, a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament was identified. Histological analysis confirmed the ligamentous nature of this structure. Furthermore, Golgi tendon organs were observed within the ligamentous structure. CONCLUSION: This is the first study showing the presence of mechanoreceptors within the ligamentous structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. The ligamentous structure could contribute to stability of the knee by providing proprioceptive input, while preservation of the ligamentous structure might ensure a better functional outcome after surgery.
Assuntos
Ligamento Cruzado Anterior/citologia , Mecanorreceptores , Meniscos Tibiais/citologia , Idoso , Ligamento Cruzado Anterior/inervação , Lesões do Ligamento Cruzado Anterior/epidemiologia , Cadáver , Humanos , Traumatismos do Joelho/epidemiologia , Articulação do Joelho , Masculino , Meniscos Tibiais/inervação , PropriocepçãoRESUMO
Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.
Assuntos
Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/metabolismo , Engenharia Tecidual/métodos , Humanos , Poliésteres/química , Alicerces Teciduais/químicaRESUMO
Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2-108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I (< 6 weeks), II (7-12 weeks), III (13-20 weeks), IV (21-50 weeks) and V (> 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p < 0.05) except for the presence of ovoid fibroblast cells (p = 0.53). Furthermore, number of myofibroblasts and cells with Golgi apparatus and rough endoplasmic reticulum was seen to peak at 13-20 weeks following injury. We describe ultrastructural features of fibroblast of different morphology along with myofibroblasts in the ligaments following injury, the changes in which might have a potential bearing on ligament healing.
Assuntos
Lesões do Ligamento Cruzado Anterior/patologia , Ligamento Cruzado Anterior/ultraestrutura , Tíbia/ultraestrutura , Adolescente , Adulto , Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/patologia , Ligamento Cruzado Anterior/cirurgia , Lesões do Ligamento Cruzado Anterior/cirurgia , Artroscopia , Retículo Endoplasmático Rugoso/ultraestrutura , Feminino , Complexo de Golgi/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Miofibroblastos/citologia , Miofibroblastos/ultraestrutura , Estudos Prospectivos , Tíbia/citologia , Tíbia/patologia , Tíbia/cirurgia , Fatores de Tempo , Adulto JovemRESUMO
With the growing number of anterior cruciate ligament (ACL) ruptures and the increased interest for regenerative medicine procedures, many studies are now concentrated on developing bioactive and biodegradable synthetic ligaments. For this application, the choice of raw materials with appropriate physicochemical characteristics and long-term degradation features is essential. Polycaprolactone (PCL) has the advantage of slow degradation that depends on its molecular weight. This study evaluates two PCL materials: a technical grade (PC60: 60 kDa) versus a medical grade (PC12: 80 kDa), both before and after functionalization with poly(sodium styrene sulfonate) (pNaSS). After determining the grafting process had little to no effect on the PCL physicochemical properties, sheep ACL fibroblast responses were investigated. The PC12 films induced a significantly lower expression of the tumor necrosis factor alpha inflammatory gene compared to the PC60 films. Both film types induced an overproduction of fibroblast growth factor-2 and transforming growth factor beta compared to the controls on day 5 and demonstrated collagen gene expression profiles similar to the controls on day 7. Upon protein adsorption, pNaSS grafting caused a rapid cell adhesion in the first 30 min and an increased adhesion strength (1.5-fold higher). Moreover, after 7 days, an increase in cell density and actin network development were noted on the grafted films.
Assuntos
Ligamento Cruzado Anterior/citologia , Materiais Biocompatíveis/toxicidade , Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Poliésteres/toxicidade , Poliestirenos/toxicidade , Animais , Materiais Biocompatíveis/química , Linhagem Celular , Fenômenos Químicos , Citocinas/metabolismo , Poliésteres/química , Poliestirenos/química , OvinosRESUMO
OBJECTIVE: To measure the density of cellular phenotypes in canine caudal cruciate ligament (CaCL), cranial cruciate ligament (CrCL), medial collateral ligament (MCL), and long digital extensor tendon (LDET). STUDY DESIGN: Ex-vivo study. METHODS: Ten CaCL, CrCL, MCL, and LDET obtained from 1 stifle of 10 dogs with no gross pathology were analyzed histologically. The density of cells with 3 nuclear phenotypes (fusiform, ovoid, and spheroid) was determined within the core region of each specimen. RESULTS: Cells with fusiform nuclei were most dense in the MCL (median [range], 319 [118-538] cells/mm2 ) and LDET (331 [61-463]), whereas cells with ovoid nuclei were most dense in the CaCL (276 [123-368]) and CrCL (212 [165-420]). The spheroid nuclear phenotype had the lowest density in all structures (31 [5-61] in CaCL, 54 [5-90] in CrCL, 2 [0-14] in MCL, and 5 [0-80] in LDET); however, the CrCL contained a denser population of spheroid cells compared with MCL and LDET (P < .05). Total cell densities did not differ among the 4 structures (P > .05). CONCLUSION: Phenotype density varied within the ligaments and tendon tested here. The cell population of CaCL and CrCL differed from that of dense collagenous tissues such as MCL and LDET. CLINICAL SIGNIFICANCE: The relatively higher density of spheroid phenotype in CrCL may reflect a distinctive native cellular population or a cellular transformation secondary to unique mechanical environment or hypoxia. This intrinsic cellular population may explain altered tissue properties prone to pathological rupture or poor healing potential of the canine CrCL.
Assuntos
Ligamento Cruzado Anterior/citologia , Cães/anatomia & histologia , Joelho de Quadrúpedes/anatomia & histologia , Tendões/citologia , Animais , Lesões do Ligamento Cruzado Anterior/veterinária , Fenômenos Biomecânicos , Fenótipo , Ruptura/veterinária , Joelho de Quadrúpedes/fisiologia , TíbiaRESUMO
Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.-Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.
Assuntos
Ligamento Cruzado Anterior/metabolismo , Moléculas de Adesão Celular/biossíntese , Regulação da Expressão Gênica , Células-Tronco/metabolismo , Adolescente , Adulto , Ligamento Cruzado Anterior/citologia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Condrócitos , Feminino , Humanos , Masculino , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Células-Tronco/citologiaRESUMO
The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.
Assuntos
Tecido Adiposo/metabolismo , Lesões do Ligamento Cruzado Anterior/patologia , Articulação do Joelho/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/patologia , Patela/citologia , Tecido Adiposo/citologia , Adulto , Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/patologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Transcriptoma , Adulto JovemRESUMO
This study aims to confirm the effects of synoviocytes (SCs) on regulating lysyl oxidases (LOXs) and matrix metalloproteinase (MMP)-1, 2, 3 in the normal and injured anterior cruciate ligament (ACL) fibroblasts response to tumor necrosis factor-α(TNF-α). The gene and protein expression levels of LOXs and MMP-1, 2, 3 in SCs cocultured ACL fibroblasts (ACLfs) induced by TNF-α and mechanical injury were analyzed by real-time polymerase chain reaction (PCR) and western bolting; the MMP-2 activity were analyzed by zymography. The results exhibited that TNF-α alone slightly downregulated the expressions of LOXs and upregulated the expression of MMP-1, 2, 3 in both normal and injured ACL fibroblasts. The decrease of LOXs and increase of MMP-1, 2, 3 in ACLfs response to TNF-α were further promoted by coculture. Taken together, these results show for the first time that the crosstalk between ACLfs and SCs could modulate the LOXs and MMP-1, 2, 3 synthesis in ACLfs in the presence of TNF-α. Accumulation of MMPs in the isolated fluid-containing space not only disrupts the balance of ACL healing, but also increases cartilage degradation and accelerates osteoarthritis (OA) in injured joint. Based on this mechanism, targeting inhibition of MMPs could provide a promising therapeutic strategy for acute ligament injury.
Assuntos
Ligamento Cruzado Anterior/citologia , Fibroblastos/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Células Cultivadas , Técnicas de Cocultura , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regulação para CimaRESUMO
Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.
Assuntos
Ligamento Cruzado Anterior/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Consumo de Oxigênio , Oxigênio/farmacologia , Adulto , Apoptose , Caspases , Sobrevivência Celular , Feminino , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/genética , Pessoa de Meia-Idade , Fosfotransferases , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Objective Osteoarthritis (OA) is induced by accumulated mechanical stress to joints; however, little has been reported regarding the cause among detailed mechanical stress on cartilage degeneration. This study investigated the influence of the control of abnormal joint movement induced by anterior cruciate ligament (ACL) injury in the articular cartilage. Design The animals were divided into 3 experimental groups: CAJM group ( n = 22: controlling abnormal joint movement), ACL-T group ( n = 22: ACL transection or knee anterior instability increased), and INTACT group ( n = 12: no surgery). After 2 and 4 weeks, the knees were harvested for digital microscopic observation, soft X-ray analysis, histological analysis, and synovial membrane molecular evaluation. Results The 4-week OARSI scores showed that cartilage degeneration was significantly inhibited in the CAJM group as compared with the ACL-T group ( P < 0.001). At 4 weeks, the osteophyte formation had also significantly increased in the ACL-T group ( P < 0.001). These results reflected the microscopic scoring and soft X-ray analysis findings at 4 weeks. Real-time synovial membrane polymerase chain reaction analysis for evaluation of the osteophyte formation-associated factors showed that the mRNA expression of BMP-2 and VEGF in the ACL-T group had significantly increased after 2 weeks. Conclusions Typically, abnormal mechanical stress induces osteophyte formation; however, our results demonstrated that CAJM group inhibited osteophyte formation. Therefore, controlling abnormal joint movement may be a beneficial precautionary measure for OA progression in the future.
Assuntos
Lesões do Ligamento Cruzado Anterior/fisiopatologia , Ligamento Cruzado Anterior/citologia , Instabilidade Articular/fisiopatologia , Articulação do Joelho/fisiopatologia , Osteófito/fisiopatologia , Animais , Cartilagem Articular/fisiopatologia , Modelos Animais de Doenças , Osteoartrite do Joelho/fisiopatologia , Ratos , Ratos WistarRESUMO
Intra-articular ligamentous injuries are typically unrepairable and have limited outcomes after graft reconstruction. A combination of porous polycaprolactone fumarate (PCLF) scaffolds with polyethylene terephthalate (PET) sutures was developed with the goal of regenerating intra-articular ligaments. Scaffolds were fabricated by injecting PCLF over three-dimensional-printed molds containing two strands of PET suture down its central pore followed by cross-linking. Scaffolds were seeded with human mesenchymal stem cells (MSCs) from adipose tissue. To demonstrate cell attachment and proliferation in culture, we performed live/dead staining and cell proliferation assays. These experiments showed that MSCs remain viable and continue to proliferate on the scaffolds in culture for at least 2 weeks. Bare scaffolds were then used to reconstruct the rabbit anterior-cruciate ligament (ACL), while control rabbits underwent semitendinosus autograft reconstruction. The specimens underwent micro-computed tomography (CT) imaging, histological examination, and biomechanical testing at 8 weeks. The ultimate pull-out strength of the PCLF-PET scaffolds and tendon autografts was initially 72 ± 30 N and to 45 ± 10 N, respectively (p < 0.06). On inspection after 8 weeks in vivo, the intra-articular portion of the PCLF-PET scaffolds was fragmented while the tendon autografts remained intact. Cross-sectional areas of bone tunnels in the PCLF-PET scaffolds (11.3 ± 1 mm2) were enlarged compared to tendon autografts (3.8 ± 0.5 mm2) (p < 0.004) as measured by micro-CT. These studies show that PET-reinforced PCLF scaffolds are capable of initial ACL reconstruction and supports stem cell growth. The intra-articular portion of the scaffold may need to be re-engineered to support their use in ligament regeneration.
Assuntos
Poliésteres/química , Poliésteres/farmacologia , Polietilenotereftalatos/química , Polietilenotereftalatos/farmacologia , Alicerces Teciduais/química , Ligamento Cruzado Anterior/citologia , Reconstrução do Ligamento Cruzado Anterior/métodos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacosRESUMO
One of the ligaments most often damaged during sports-the anterior cruciate ligament (ACL)-has poor healing capacity. On damage, reconstructive surgery is performed to restore the mechanical stability of the knee and to reduce the inflammatory milieu otherwise present in the joint. A return to normal activities, however, takes between 9 and 12 months. Thus, strategies capable of improving ACL graft healing are needed. Embryonic development of tendon and ligament (T/L) is regulated by a crosstalk between different cell types. We hypothesized that terminally differentiated skeletal-derived cells such as osteoblasts, chondrocytes, and myoblasts modulate T/L healing. Using an indirect coculture system, we discovered that myoblast-secreted signals-but not osteoblasts, chondrocytes, or stromal-secreted signals-are capable of upregulating classical T/L markers such as scleraxis and tenomodulin on human hamstring tendon-derived cells (hTC), which contribute to ACL graft healing. Transcriptome analysis showed that coculturing hTC with myoblasts led to an upregulation of extracellular matrix (ECM) genes and resulted in enhanced ECM deposition. In vivo, using a rat model of ACL reconstruction showed that conditioned media derived from human muscle tissue accelerated femoral tunnel closure, a key step for autograft integration. Collectively, these results indicate that muscle-secreted signals can be used to improve ACL graft healing in a clinical setting where muscle remnants are often discarded.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/citologia , Cicatrização/fisiologia , Animais , Ligamento Cruzado Anterior/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados , Matriz Extracelular/metabolismo , Humanos , Masculino , Camundongos , Mioblastos/citologia , Ratos , Ratos Sprague-Dawley , Tendões/citologiaRESUMO
Anterior cruciate ligament injuries are common in humans, though cellular components of the knee have little regenerative or proliferation potential. This study investigated the differentiation of human amnion-derived mesenchymal stem cells (hAMSCs) into human anterior cruciate ligament fibroblasts (hACLFs) in vitro through induction with bFGF and TGF-ß1 with coculture systems. Groups A and B comprised hAMSCs at the 3rd passage cultured with and without bFGF and TGF-ß1, respectively; Groups C and D consisted of hAMSCs and hACLFs in monolayer coculture with and without bFGF and TGF-ß1, respectively; Groups E and F were composed of hAMSCs and hACLFs in Transwell coculture with and without bFGF and TGF-ß1, respectively. Cell morphology and proliferation were recorded. Protein expression and relative mRNA expression were evaluated in each group. Cell proliferation was significantly higher in the induced groups than in the noninduced groups. Protein expression increased over time with the highest expression observed in Group E. mRNA levels were significantly higher in Group E than in other groups. This study is the first to demonstrate the use of the Transwell coculture system for this purpose, and hAMSCs were successfully differentiated into hACLFs. Thus, hAMSCs may be a superior choice for hACLF differentiation via Transwell coculture.
Assuntos
Âmnio/metabolismo , Ligamento Cruzado Anterior/metabolismo , Diferenciação Celular , Fibroblastos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Âmnio/citologia , Ligamento Cruzado Anterior/citologia , Técnicas de Cocultura , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Attempts have been made to validate the significance of remnant preservation with anterior cruciate ligament (ACL) reconstruction using immunohistochemical and immunocytochemical techniques. The purpose of this study was to examine the expression of mechanoreceptors in the remnant tissue of ACL reconstruction performed with the remnant-preserving technique. METHODS: Tissue samples were obtained from 10 patients who underwent ACL reconstruction with the remnant-preserving technique. The specimens were obtained from remnant ACL tissue and Achilles allografts superficially and at the tibial attachment. The control group consisted of three normal ACLs procured from young males who underwent partial meniscectomy. Tissues and cells from the ACL remnants and Achilles allografts were characterized using hematoxylin and eosin (H&E) staining and immunohistochemical, immunocytochemical, and immunoblotting assays. In particular, the sensitivity of neural cell validation was improved using nerve growth factor (NGF) to stimulate the expression of neural cells. RESULTS: The results are summarized as follows. (1) In H&E staining and immunohistochemical assays, no neural cells were detected in remnant or allograft tissue. (2) In the immunocytochemical study, neural cells were detected in remnant tissue. (3) The increased proliferation of remnant ACL cells with NGF treatment suggested their identity as neural cells. (4) NGF treatment also stimulated protein and RNA expression of Nestin (a specific marker for neural cells) in remnant ACL cells. CONCLUSIONS: The improved immunocytochemical methodology proved useful. Although mechanoreceptors were detected relatively less frequently than expected, the authors consider that this finding does not negate the necessity of remnant-preserving ACL reconstruction.
Assuntos
Tendão do Calcâneo/transplante , Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/citologia , Mecanorreceptores , Adolescente , Adulto , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissue have the potential for high proliferation and multilineage differentiation that can accelerate tendon-bone healing. While patient characteristics, such as age, can affect tendon-bone healing, the influence of elapsed time after injury on the healing process is unclear. HYPOTHESIS: Cells obtained during the early phase after injury will exhibit a greater tendon-bone healing potential compared with chronic phase counterparts when applied to an immunodeficient rat model of ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: Adult human ACL-ruptured tissue was harvested from patients undergoing arthroscopic primary ACL reconstruction and classified into 2 groups based on the time elapsed between injury and surgery: (1) early group (≤3 months from injury) and (2) chronic group (>3 months from injury). In addition, 76 ten-week-old female immunodeficient rats underwent ACL reconstruction, followed by intracapsular administration of one of the following: (1) ACL-derived cells from the early group (n = 5), (2) ACL-derived cells from the chronic group (n = 5), or (3) phosphate-buffered saline (PBS) only (n = 5). During the 8 weeks after surgery, histological (weeks 2, 4, 8), immunohistochemical (week 2), radiographic (weeks 0, 2, 4, 8), and biomechanical (week 8) analyses were performed to evaluate tendon-bone healing. RESULTS: In the early group, the histological evaluation showed early healing, induction of endochondral ossification-like integration, and mature bone ingrowth. Micro-computed tomography showed that the tibial bone tunnels at week 4 and week 8 were significantly reduced in the early group compared with those in the chronic group and PBS group ( P < .05). Moreover, biomechanical tensile strength was significantly greater in the early group than in the other groups ( P < .05). An accelerated healing potential in the early group was further demonstrated by the enhancement of intrinsic angiogenesis/osteogenesis and human-derived vasculogenesis/osteogenesis. CONCLUSION: Compared with human ACL-derived cells obtained during the chronic phase, cells obtained during the early phase after injury have a greater tendon-bone healing potential when used in an immunodeficient rat model of ACL reconstruction. CLINICAL RELEVANCE: During ACL reconstruction surgery, transplanting ACL remnant tissue in the early phase after injury could accelerate and enhance tendon-bone healing.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/citologia , Cicatrização/fisiologia , Adulto , Animais , Ligamento Cruzado Anterior/cirurgia , Diferenciação Celular , Proliferação de Células , Feminino , Humanos , Osteogênese , Ratos Nus , Tendões/transplante , Resistência à Tração , Tíbia/diagnóstico por imagem , Tíbia/fisiologia , Tíbia/cirurgia , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Anterior cruciate ligament (ACL) reconstruction remains a formidable clinical challenge because of the lack of vascularization and adequate cell numbers in the joint cavity. In this study, we developed a novel strategy to mimic the early stage of repair in vivo, which recapitulated extra-articular inflammatory response to facilitate the early ingrowth of blood vessels and cells. A vascularized ectopic tissue engineered ligament (ETEL) with silk collagen scaffold was developed and then transferred to reconstruct the ACL in rabbits without interruption of perfusion. At 2weeks after ACL reconstruction, more well-perfused cells and vessels were found in the regenerated ACL with ETEL, which decreased dramatically at the 4 and 12week time points with collagen deposition and maturation. ACL treated with ETEL exhibited more mature ligament structure and enhanced ligament-bone healing post-reconstructive surgery at 4 and 12weeks, as compared with the control group. In addition, the ETEL group was demonstrated to have higher modulus and stiffness than the control group significantly at 12weeks post-reconstructive surgery. In conclusion, our results demonstrated that the ETEL can provide sufficient vascularity and cellularity during the early stages of healing, and subsequently promote ACL regeneration and ligament-bone healing, suggesting its clinic use as a promising therapeutic modality. STATEMENT OF SIGNIFICANCE: Early inflammatory cell infiltration, tissue and vessels ingrowth were significantly higher in the extra-articular implanted scaffolds than theses in the joint cavity. By mimicking the early stages of wound repair, which provided extra-articular inflammatory stimulation to facilitate the early ingrowth of blood vessels and cells, a vascularized ectopic tissue engineered ligament (ETEL) with silk collagen scaffold was constructed by subcutaneous implantation for 2weeks. The fully vascularized TE ligament was then transferred to rebuild ACL without blood perfusion interruption, and was demonstrated to exhibit improved ACL regeneration, bone tunnel healing and mechanical properties.
Assuntos
Lesões do Ligamento Cruzado Anterior/terapia , Reconstrução do Ligamento Cruzado Anterior/instrumentação , Ligamento Cruzado Anterior/transplante , Órgãos Bioartificiais , Colágeno/química , Seda/química , Alicerces Teciduais , Animais , Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/crescimento & desenvolvimento , Lesões do Ligamento Cruzado Anterior/patologia , Lesões do Ligamento Cruzado Anterior/fisiopatologia , Reconstrução do Ligamento Cruzado Anterior/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Projetos Piloto , Coelhos , Regeneração/fisiologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Resultado do TratamentoRESUMO
Anterior cruciate ligament (ACL) ruptures reconstructed with tendon grafts are commonly fixed with bioabsorbable implants, which are frequently complicated by incomplete bone filling upon degradation. Bone regeneration after ACL reconstruction could be enhanced by utilizing tissue engineering techniques and three-dimensional (3D) printing to create a porous bioabsorbable scaffold with delayed delivery of recombinant-human bone morphogenetic protein 2 (rhBMP-2). The first aim of this study was to design a 3D poly(propylene fumarate) (PPF) porous scaffold that maintained suitable pullout strength for future testing in a rabbit ACL reconstruction model. Our second aim was to determine the release kinetics of rhBMP-2 from PPF scaffolds that utilized both calcium-phosphate coatings and growth factor delivery on microspheres, both of which have been shown to decrease the initial burst release of rhBMP-2 and increase bone regeneration. To determine the degree of scaffold porosity that maintained suitable pullout strength, tapered scaffolds were fabricated with increasing porosity (0%, 20%, 35%, and 44%) and pullout testing was performed in a cadaveric rabbit ACL reconstruction model. Scaffolds were coated with carbonate hydroxyapatite (synthetic bone mineral [SBM]), and radiolabeled rhBMP-2 was delivered in four different experimental groups as follows: Poly(lactic-co-glycolic acid) microspheres only, microspheres and collagen (50:50), collagen only, and saline solution only. rhBMP-2 release was measured at day 1, 2, 4, 8, 16, and 32. The microsphere delivery groups had a smaller burst release and released a smaller percentage of rhBMP-2 over the 32 days than the collagen and saline only groups. In conclusion, a porous bioabsorbable scaffold with suitable strength for a rabbit ACL reconstruction was developed. Combining a synthetic bone mineral coating with microspheres had an additive effect, decreasing the initial burst release and cumulative release of rhBMP-2. Future studies need to evaluate this scaffold's fixation strength and bone filling capabilities in vivo compared to traditional bioabsorbable implants.
Assuntos
Ligamento Cruzado Anterior/citologia , Proteína Morfogenética Óssea 2/química , Fumaratos/química , Polipropilenos/química , Alicerces Teciduais/química , Fator de Crescimento Transformador beta/química , Animais , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
PURPOSES: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2 to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. MATERIALS AND METHODS: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. RESULTS: The real-time PCR results show that mechanical stretch or CoCl2 treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2 treatment group alone. Western blot analysis and ELISA also confirmed these results. CONCLUSIONS: Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.
Assuntos
Ligamento Cruzado Anterior/metabolismo , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Estresse Mecânico , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto , Ligamento Cruzado Anterior/citologia , Células Cultivadas , Cobalto/farmacologia , Feminino , Fibroblastos/citologia , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Strong graft-bone integration is a prerequisite for successful graft remodeling after reconstruction of the anterior cruciate ligament (ACL) using soft tissue grafts. Novel strategies to accelerate soft tissue graft-bone integration are needed to reduce the need for bone-tendon-bone graft harvest, reduce patient convalescence, facilitate rehabilitation, and reduce total recovery time after ACL reconstruction. HYPOTHESIS: The application of ACL-derived stem cells with enhanced expression of bone morphogenetic protein 2 (BMP2) onto soft tissue grafts in the form of cell sheets will both accelerate and improve the quality of graft-bone integration after ACL reconstruction in a rat model. STUDY DESIGN: Controlled laboratory study. METHODS: ACL-derived CD34+ cells were isolated from remnant human ACL tissues, virally transduced to express BMP2, and embedded within cell sheets. In a rat model of ACL injury, bilateral single-bundle ACL reconstructions were performed, in which cell sheets were wrapped around tendon autografts before reconstruction. Four groups containing a total of 48 rats (96 knees) were established (n = 12 rats; 24 knees per group): CD34+BMP2 (100%), CD34+BMP2 (25%), CD34+ (untransduced), and a control group containing no cells. Six rats from each group were euthanized 2 and 4 weeks after surgery, and each graft was harvested for immunohistochemical and histological analyses. The remaining 6 rats in each group were euthanized at 4 and 8 weeks to evaluate in situ tensile load to failure in each femur-graft-tibia complex. RESULTS: In vitro, BMP2 transduction promoted the osteogenic differentiation of ACL-derived CD34+ cells while retaining their intrinsic multipotent capabilities. Osteoblast densities were greatest in the BMP2 (100%) and BMP2 (25%) groups. Bone tunnels in the CD34+BMP2 (100%) and CD34+BMP2 (25%) groups had the smallest cross-sectional areas according to micro-computed tomography analyses. Graft-bone integration occurred most rapidly in the CD34+BMP2 (25%) group. Tensile load to failure was significantly greater in the groups containing stem cells at 4 and 8 weeks after surgery. Tensile strength was greatest in the CD34+BMP2 (100%) group at 4 weeks, and in the CD34+BMP2 (25%) group at 8 weeks. CONCLUSION: ACL-derived CD34+ cells transduced with BMP2 accelerated graft-bone integration after ACL reconstruction using soft tissue autografts in a rat model, as evidenced by improved histological appearance and graft-bone interface biology along with tensile load to failure at each time point up to 8 weeks after surgery. CLINICAL RELEVANCE: A primary disadvantage of using soft tissue grafts for ACL reconstruction is the prolonged time required for bony ingrowth, which delays the initiation of midsubstance graft remodeling. The lack of consistent correlation between the appearance of a "healed" ACL on postoperative magnetic resonance imaging and readiness to return to sport results in athletes being released to sport before the graft is ready to handle high-intensity loading. Therefore, it is desirable to identify strategies that accelerate graft-bone integration, which would reduce the time to biologic fixation, improve the reliability of biologic fixation, allow for accelerated rehabilitation, and potentially reduce the incidence of early graft pullout and late midsubstance failure.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/citologia , Proteína Morfogenética Óssea 2/metabolismo , Osteogênese , Transplante de Células-Tronco , Adolescente , Adulto , Animais , Diferenciação Celular , Feminino , Humanos , Ratos , Ratos Nus , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Objective: To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from rats in vitro. Methods: Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 (CCK-8), and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas (ALP), bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 (BMP-2), secreted phosphoprotein 1 (Spp1), collagen type II α1 (Col2α1), Aggrecan (Acan), Sox9, peroxisome proliferator activated receptor γ2 (PPARγ2), and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR. Results: BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance ( A) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) ( t=3.599, P=0.023); the number of colonies of ACL-MSCs [(53.00±5.51)/hole] was significantly more than that of BMSCs [(30.67±4.84)/hole] ( t=3.045, P=0.038). The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs ( P<0.01). Conclusion: The proliferation potential of ACL-MSCs is greater than that of BMSCs, and the former is apt to differentiate into chondrocytes. ACL-MSCs are promising cells to promote tendon-bone healing.