Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury.

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.

Ultrastructural and histological changes in tibial remnant of ruptured anterior cruciate ligament stumps: a transmission electron microscopy and immunochemistry-based observational study.

OBJECTIVES: Anterior cruciate ligament (ACL) rupture is a common injury and has a non-union rate of 40-100%. Important cellular events, such as fibroblast proliferation, angiogenesis and change in collagen fibril thickness in the ACL remnant, as described in other dense connective tissue, might have an implication in graft recovery following ACL reconstruction. Thus we conducted a study with an aim to characterize the ultrastructural and histological features of ruptured ACL tibial stump and correlate the same with the duration of injury. MATERIALS AND METHODS: This was a prospective observational study in which 60 ruptured human ACLs were evaluated for collagen fibril thickness, blood vessel density (per mm2) and fibroblast density (per mm2) with the help of transmission electron microscopy, immunohistochemistry via CD34 antibody staining and light microscopy (H&E staining). The findings were correlated with duration of injury. RESULTS: Fifty-four male and six female patients with a mean duration of the injury of 23.01 weeks (SD = 26.09; range 2-108 weeks) were included for the study and were divided on the basis of duration of injury as follows: Group I (≤ 6 weeks; N = 16), Group II (7-12 weeks; N = 18), Group III (13-20 weeks; N = 7), Group IV (21-50 weeks; N = 12), Group V (> 50 weeks; N = 7). A significant correlation was seen with blood vessel density (r = 0.303, p = 0.01) and fibroblast density (r = - 0.503, p = 0.001). Thickness of collagen fibril did not correlate with the duration of injury (r = 0.15, p = 0.23). The thickness of the collagen reached its peak after 50 weeks following injury, whereas highest density of blood vessel and fibroblast was seen at 12-20 weeks. Matched pair analysis revealed a significant decrease in collagen fibril thickness and an increase in fibroblast density at 7-12 weeks. CONCLUSION: Following injury to ACL, the ruptured tibial stump undergoes a series of changes at the cellular level vis-à-vis changes in collagen fibril thickness, vascular density and fibroblast density that possibly suggest an intrinsic healing response. This further may have implications on the functional outcome following ACL reconstruction with remnant preservation. LEVEL OF EVIDENCE: III.

Ultrastructure and three-dimensional architecture of the anterior cruciate ligament in the knee joints of young and old monkeys.

We examined the ultrastructure of the anterior cruciate ligament and assessed age-related changes by comparing the ligaments of young and old monkeys. Ultrathin sections of the anterior cruciate ligament were observed by transmission electron microscopy. The three-dimensional architecture of collagen fibers in the ligament was examined by scanning electron microscopy after tissue specimens were treated with 2 N NaOH to digest the extracellular matrix. At the surface layer of the cruciate ligament in young monkeys, fusiform-shaped fibroblasts actively produced collagen fibrils. The ligament consisted of parallel bundles of dense collagen fibrils of approximately 200 nm in diameter. Collagen fibrils appeared to run linearly. Ligament fibrocytes in the deep layer had a stellate form. Ligament fibrocytes decreased in number and showed marked atrophy in old age. Collagen fibrils had a looser configuration in older monkeys. Despite atrophy of fibroblasts in the deep layer of the anterior cruciate ligament, the area with atrophic fibroblasts in the ligament expands with age, which can likely cause deterioration of and a reduction in collagen fibers. This information can be applied in studies on the cause of the low repair ability of and aging-related changes in the anterior cruciate ligament in humans.

Overlap Between Anterior Cruciate Ligament and Anterolateral Meniscal Root Insertions: A Scanning Electron Microscopy Study.

BACKGROUND: The anterolateral meniscal root (ALMR) has been reported to intricately insert underneath the tibial insertion of the anterior cruciate ligament (ACL). Previous studies have begun to evaluate the relationship between the insertion areas and the risk of iatrogenic injuries; however, the overlap of the insertions has yet to be quantified in the sagittal and coronal planes. PURPOSE: To investigate the insertions of the human tibial ACL and ALMR using scanning electron microscopy (SEM) and to quantify the overlap of the ALMR insertion in the coronal and sagittal planes. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten cadaveric knees were dissected to isolate the tibial ACL and ALMR insertions. Specimens were prepared and imaged in the coronal and sagittal planes. After imaging, fiber directions were examined to identify the insertions and used to calculate the percentage of the ACL that overlaps with the ALMR instead of inserting into bone. RESULTS: Four-phase insertion fibers of the tibial ACL were identified directly medial to the ALMR insertion as they attached onto the tibial plateau. The mean percentage of ACL fibers overlapping the ALMR insertion instead of inserting into subchondral bone in the coronal and sagittal planes was 41.0% ± 8.9% and 53.9% ± 4.3%, respectively. The percentage of insertion overlap in the sagittal plane was significantly higher than in the coronal plane ( P = .02). CONCLUSION: This study is the first to quantify the ACL insertion overlap of the ALMR insertion in the coronal and sagittal planes, which supplements previous literature on the insertion area overlap and iatrogenic injuries of the ALMR insertion. Future studies should determine how much damage to the ALMR insertion is acceptable to properly restore ACL function without increasing the risk for tears of the ALMR. CLINICAL RELEVANCE: Overlap of the insertion areas on the tibial plateau has been previously reported; however, the results of this study demonstrate significant overlap of the insertions superior to the insertion sites on the tibial plateau as well. These findings need to be considered when positioning for tibial tunnel creation in ACL reconstruction to avoid damage to the ALMR insertion.

Ultrastructure of the three anterior cruciate ligament bundles.

The anterior cruciate ligament (ACL) can be morphologically separated into not only two, but three bundles: the anteromedial-medial bundle (AM-MB), the anteromedial-lateral bundle (AM-LB), and the posterolateral bundle (PLB). Our hypothesis was that the three bundles differ in their microstructures. The purpose of this study was to clarify the microstructural differences among the three bundles. The normal ACLs of six fresh frozen cadavers were harvested. After the AM-MB, AM-LB, and PLB were identified, their fibril structures were analyzed using a transmission electron microscope. The fibril orientation, distribution pattern, and the mass average diameter of the fibrils (MAD) were compared among the AM-MBs, AM-LBs, and PLBs. The AM-MB and AM-LB fibrils were arranged mostly in the longitudinal direction, while the PLB fibrils were not aligned in a uniform direction. The fibril diameter distribution pattern of AM-MBs showed a bi-modal pattern due to the existence of small-diameter (30-40 nm) and large-diameter fibrils (70-80 nm), while that of the AM-LBs and PLBs had a unimodal pattern with one prominent high peak at a diameter of 50-60 nm. The mean MAD of the AM-MBs (83.2 - 11.2 nm) was significantly larger than that of the PLBs (66.8 - 7.7 nm), while it showed no significant difference compared to that of the AM-LBs (77.6 - 12.3 nm). The three ACL bundles have different ultrastructures. The AM-MB predominantly includes thick, uni-directionally oriented fibrils like tendons, while the PLB consists of thinner, multi-directionally oriented fibrils. The AM-LB shows an intermediate structure between the AM-MB and the PLB.

Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds.

Reconstituted extracellular matrix (ECM)-derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but does not harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function, low-temperature ethylene oxide and 15 kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium, and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament, and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials.

Crimp morphology in the ovine anterior cruciate ligament.

While the crimp morphology in ligaments and tendons has been described in detail in the literature, its relative distribution within the tissue has not been studied, especially in relation to the complex multi-bundle arrangement as is found in the anterior cruciate ligament (ACL). In this study, the crimp morphology of the ovine ACL was examined topologically and with respect to its double-bundle structure. The crimp morphologies were compared with the knee in three knee positions, namely stance, maximum extension and maximum flexion. As a control, the crimp morphology of the ACL free from its bony attachments was determined. In the control samples, the anterior-medial (AM) bundle contained a combination of coarse and fine crimp, whereas the posterior-lateral (PL) bundle manifested only a coarse crimp. Using the extent of crimp loss observed when subjecting the knee to the respective positions, and comparing with the controls, the crimp morphologies show that the AM bundle of the ACL is most active in the stance position, whereas for the maximum extension and flexion positions the PL bundle is most active. We propose that these differences in crimp morphologies have relevance to ACL design and function.

A multi-scale structural study of the porcine anterior cruciate ligament tibial enthesis.

Like the human anterior cruciate ligament (ACL), the porcine ACL also has a double bundle structure and several biomechanical studies using this model have been carried out to show the differential effect of these two bundles on macro-level knee joint function. It is hypothesised that if the different bundles of the porcine ACL are mechanically distinct in function, then a multi-scale anatomical characterisation of their individual enthesis will also reveal significant differences in structure between the bundles. Twenty-two porcine knee joints were cleared of their musculature to expose the intact ACL following which ligament-bone samples were obtained. The samples were fixed in formalin followed by decalcification with formic acid. Thin sections containing the ligament insertion into the tibia were then obtained by cryosectioning and analysed using differential interference contrast (DIC) optical microscopy and scanning electron microscopy (SEM). At the micro-level, the anteromedial (AM) bundle insertion at the tibia displayed a significant deep-rooted interdigitation into bone, while for the posterolateral (PL) bundle the fibre insertions were less distributed and more focal. Three sub-types of enthesis were identified in the ACL and related to (i) bundle type, (ii) positional aspect within the insertion, and (iii) specific bundle function. At the nano-level the fibrils of the AM bundle were significantly larger than those in the PL bundle. The modes by which the AM and PL fibrils merged with the bone matrix fibrils were significantly different. A biomechanical interpretation of the data suggests that the porcine ACL enthesis is a specialized, functionally graded structural continuum, adapted at the micro-to-nano scales to serve joint function at the macro level.

Intrinsic healing response of the human anterior cruciate ligament: an histological study of reattached ACL remnants.

A reattachment of the tibial remnant of the torn anterior cruciate ligament (ACL) to the posterior cruciate ligament is sometimes observed during surgery and apparently implies that the human ACL does have a healing response. The aim of this study was to investigate whether this reattachment tissue has similar histological characteristics of a healing response as the medial collateral ligament (MCL), which can heal spontaneously. Standard histology and immunostaining of α-smooth muscle actin and collagen type 3 was performed. The results shows that the reattached tissue has typical characteristics of a healing response: there attached ACL remnant could not be released by forceful traction; microscopy showed that the collagen fibers of the reattached tissue were disorganized with no preferred direction; increased neovascularization; the presence of lipid vacuoles; the mean number of cells within the biopsy tissue was 631±269 cells per mm2; and 68±20% was expressing α-SMA; semi-quantitative analysis of collagen type 3 expression showed that collagen type 3 had an high expression with an average score of 3. In conclusion, this study shows that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to the MCL.

Effect of mechanical strain on the collagen VI pericellular matrix in anterior cruciate ligament fibroblasts.

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.

The effect of polystyrene sodium sulfonate grafting on polyethylene terephthalate artificial ligaments on in vitro mineralisation and in vivo bone tissue integration.

This study investigates the impact of polystyrene sodium sulfonate (PolyNaSS) grafting onto the osseo-integration of a polyethylene terephthalate artificial ligament (Ligament Advanced Reinforcement System, LARS™) used for Anterior Cruciate Ligament (ACL). The performance of grafted and non-grafted ligaments was assessed in vitro by culturing human osteoblasts under osteogenic induction and this demonstrated that the surface modification was capable of up-regulating the secretion of ALP and induced higher level of mineralisation as measured 6 weeks post-seeding by Micro-Computed Tomography. Grafted and non-grafted LARS™ were subsequently implanted in an ovine model for ACL reconstruction and the ligament-to-bone interface was evaluated by histology and biomechanical testings 3 and 12 months post-implantation. The grafted ligaments exhibited more frequent direct ligament-to-bone contact and bone formation in the core of the ligament at the later time point than the non-grafted specimens, the grafting also significantly reduced the fibrous encapsulation of the ligament 12 months post-implantation. However, this improved osseo-integration was not translated into a significant increase in the biomechanical pull-out loads. These results provide evidences that PolyNaSS grafting improved the osseo-integration of the artificial ligament within the bone tunnels. This might positively influence the outcome of the surgical reconstructions, as higher ligament stability is believed to limit micro-movement and therefore permits earlier and enhanced healing.

Ultrastructural and morphological characteristics of human anterior cruciate ligament and hamstring tendons.

Hamstring tendons are a commonly used substitute for anterior cruciate ligament (ACL) reconstruction. Ligaments and tendons are similar in composition but the ACL is more complex than hamstring tendons in function and gross morphology, which are highly dependent on its structure and ultrastructure. The purpose of this study was to compare the morphology and ultrastructure of normal human ACL and hamstring tendons, including the cell type and arrangement, expression level of proteoglycans, diameter, and density of collagen fibrils. Twenty semitendinosus or gracilis tendons and 20 ACL specimens were harvested from patients with ACL rupture or osteoarthritis undergoing routine total knee arthroplasty. The specimens were examined histologically and the ultrastructure was observed using scanning and transmission electron microscopy. Semitendinosus and gracilis tendons showed a homogeneous arrangement of collagen fibers and cell type. They had lower fibril density and more widely distributed fibril diameters. In the ACL, there was a more complex arrangement of collagen fibers, distribution of proteoglycans and different cell types. Electronic microscopy demonstrated a combination of parallel, helical and nonlinear networks of ACL fibrils, and fibril diameters were smaller and more nonuniform. This study compared the anatomy of normal human ACL and hamstring tendons, which may provide a standard for evaluating hamstring tendons grafts after ACL reconstruction and may facilitate the application of hamstring tendons in clinical applications.

A novel process for optimizing musculoskeletal allograft tissue to improve safety, ultrastructural properties, and cell infiltration.

BACKGROUND: This study evaluated the properties of scaffold derived from freeze-dried human Achilles tendon allograft for use in anterior cruciate ligament (ACL) reconstruction. Our hypothesis was that such an allograft could be processed using a method to remove cellular and infectious material, producing a cytocompatible, architecturally modified scaffold possessing tensile properties suitable for ACL reconstruction. METHODS: Fifty-two allografts were provided by a tissue bank. Twenty-one were used as controls to assess cellularity, DNA content, microarchitecture, porosity, cytocompatibility, and tensile properties in vitro (n = 13) and in vivo (n = 8). Thirty-one were processed to produce scaffolds that were similarly assessed for these properties in vitro (n = 23) and in vivo (n = 8). The elimination of added enveloped and nonenveloped viruses was also determined in vitro after each processing step. RESULTS: A subjective decrease in cellularity and a significant decrease in DNA content were observed in the scaffolds compared with the allografts from which they had been derived. The porosity was increased significantly, and the scaffolds were cytocompatible in vitro. Processing resulted in significantly increased elongation of the scaffolds (138% of the elongation of the unprocessed allograft) during tensile testing. No other significant differences in tensile properties were observed in vitro or in vivo. The number of infiltrating host cells and the depth to which those cells infiltrated were significantly greater in the scaffolds. No enveloped viruses and only two of 10(8) nonenveloped viruses were detected in the scaffolds after processing, corresponding to a sterility assurance level of 0.2 × 10(-7). CONCLUSIONS: Allografts were processed using a method that removed cellular and infectious material to produce a decellularized, cytocompatible, architecturally modified scaffold with tensile properties that differed minimally from those of human allograft tissue both in vitro and in vivo. The scaffold production process also resulted in an increase in porosity that led to increased cell infiltration in vivo.

ACL reconstruction using a novel hybrid scaffold composed of polyarylate fibers and collagen fibers.

The objective was to perform an initial in vivo evaluation of a novel braided hybrid polyarylate and collagen fiber scaffold for the reconstruction of the anterior cruciate ligament (ACL). The braided hybrid scaffold is composed of 75% poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate)(12,10), [p(DTD DD)] fibers and 25% type I bovine collagen fibers. The scaffold is designed to temporarily bear mechanical loads and gradually degrade as neoligament tissue is deposited. Scaffolds were electron beam sterilized and used to reconstruct the ACL in five Finnish Dorset crossed-bred sheep in this feasibility study. At 4 (n = 1) and 12 (n = 4) weeks post-op, scaffolds were retrieved and analyzed for cellular ingrowth and strength retention. There was extensive cell infiltration and vascularity, which increased with time. Tissue ingrowth occurred throughout the cross section in the midsubstance of the scaffolds. After 12 weeks all scaffolds were intact. Femur-scaffold-tibia complex (FSTC) explanted at 12 weeks had a yield load of 42 ± 22 N and a stiffness of 9 ± 3 N mm(-1). All scaffolds were well tolerated in the intraarticular space and induced tissue ingrowth, including new blood vessels, fibroblasts, inflammatory cells, and newly deposited collagen, throughout the cross section of the scaffold. Tissue ingrowth is critical to the success of a degradable scaffold for ACL reconstruction. Long-term studies in a large animal model are required to determine the efficacy of these novel hybrid scaffolds for ACL reconstruction.

Post-natal molecular adaptations in anteromedial and posterolateral bundles of the ovine anterior cruciate ligament: one structure with two parts or two distinct ligaments?

The human anterior cruciate ligament (ACL) is a composite structure of two anatomically distinct bundles: an anteromedial (AM) and posterolateral (PL) bundles. Tendons are often used as autografts for surgical reconstruction of ACL following severe injury. However, despite successful surgical reconstruction, some people experience re-rupture and later development of osteoarthritis. Understanding the structure and molecular makeup of normal ACL is essential for its optimal replacement. Reportedly the two bundles display different tensions throughout joint motion and may be fundamentally different. This study assessed the similarities and differences in ultrastructure and molecular composition of the AM and PL bundles to test the hypothesis that the two bundles of the ACL develop unique characteristics with maturation. ACLs from nine mature and six immature sheep were compared. The bundles were examined for mRNA and protein levels of collagen types I, III, V, and VI, and two proteoglycans. The fibril diameter composition of the two bundles was examined with transmission electron microscopy. Maturation does alter the molecular and structural composition of the two bundles of ACL. Although the PL band appears to mature slower than the AM band, no significant differences were detected between the bundles in the mature animals. We thus reject our hypothesis that the two ACL bundles are distinct. The two anatomically distinct bundles of the sheep ACL can be considered as two parts of one structure at maturity and material that would result in a structure of similar functionality can be used to replace each ACL bundle in the sheep.

The effects of levofloxacin on rabbit anterior cruciate ligament cells in vitro.

Articular cartilage, epiphyseal growth plate and tendons have been recognized as targets of fluoroquinolone-induced connective tissue toxicity. The effects of fluoroquinolones on ligament tissues are still unknown. The aim of this study was to investigate the effects of levofloxacin, a typical fluoroquinolone antibiotic drug, on rabbit anterior cruciate ligament (ACL) cells in vitro. Rabbit ACL cells were treated with levofloxacin at different concentrations (0, 14, 28, 56, 112 and 224 µM) and were assessed to determine the possible cytotoxic effects of levofloxacin on ACL cells. Levofloxacin, with concentrations ranging from 28 to 224 µM, induced dose-dependent ACL cell apoptosis. Characteristic markers of programmed cell death and degenerative changes were identified by electron microscopy in the ACL cells treated with 28 µM of levofloxacin. Moreover, levofloxacin significantly increased the mRNA expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 and decreased the expression of tissue inhibitors of metalloproteinase 1 (TIMP-1) in a concentration-dependent manner; TIMP-3 and collagen type I alpha 1 (Col1A1) mRNA expression was not affected. Immunocytochemical analysis indicated that levofloxacin markedly increased the expression of active caspase-3 within a concentration range of 28 to 224 µM, whereas a clear-cut decrease in Col1A1 expression was found with levofloxacin treatment concentrations of 112 and 224 µM, compared to controls. Our data suggest that levofloxacin has cytotoxic effects on ACL cells characterized by enhanced apoptosis and decreased extracellular matrix, which suggest a potential adverse effect of fluoroquinolones.

Effect of estrogen on tissue elasticity of the ligament proper in rabbit anterior cruciate ligament: measurements using scanning acoustic microscopy.

BACKGROUND: Previous epidemiological studies revealed that anterior cruciate ligament (ACL) injuries were more frequently seen in female athletes than in male athletes. To elucidate the pathogenetic roles of estrogen in ACL ruptures, the elasticity of ACL tissue was measured using a scanning acoustic microscope (SAM) in an estrogen-controlled animal model. METHODS: A total of 40 ovariectomized Japanese white rabbits were randomly divided into four groups according to the administered dose of 17beta-estradiol (groups L, M, H, and C). Injection of 17beta-estradiol was performed 1, 2, 3, and 4 weeks after surgery, and doses in groups L, M, and H were 50, 100, and 500 microg/kg, respectively. Group C received no estradiol. Only groups L, M, and C were used for current analyses because their mean serum estrogen levels were within the physiological range (groups C, L, M, and H: 37, 50, 60, and 231 pg/ml, respectively). Five weeks after ovariectomy, the lateral portion of the ligament was harvested. Specimens were fixed with 10% neutralized formalin and embedded in paraffin. Then, 10 mum thick sections were cut perpendicular to the ligament fibers for routine histological staining and measurement with SAM. RESULTS: The mean tissue sound speeds of groups C, L, and M were 1727 +/- 32, 1683 +/- 53, and 1665 +/- 63 m/s, respectively. Group M presented significantly lower tissue sound speed than group C (P = 0.021). Furthermore, a negative correlation was found between the mean serum estrogen level and mean tissue sound speed of the ACL among all animals in groups C, L, and M (r = 0.47, P = 0.016). CONCLUSION: The results of the present study indicated that estrogen altered the tissue elasticity of rabbit ACL. Estrogen may constitute one of the pathogenetic factors in ACL rupture in female athletes.

Direct anterior cruciate ligament insertion to the femur assessed by histology and 3-dimensional volume-rendered computed tomography.

PURPOSE: The purpose of this study was to histologically identify the direct and indirect insertion of the femoral anterior cruciate ligament (ACL) insertion. Furthermore, we quantitatively measured the direct femoral insertion area by use of the 3-dimensional (3D) volume-rendered (VR) computed tomography (CT) model. METHODS: By use of 8 intact cadaveric knees, the lateral femoral condyle including the ACL attachment was sectioned for histologic examination in 3 oblique-axial planes parallel to the roof of the intercondylar notch and in the sagittal planes. Before sectioning, these knees had been subjected to CT to obtain 3D VR images of the femur. Once the direct insertion of the ACL was identified on each histologic section, the corresponding image was superimposed on the corresponding CT image. RESULTS: The direct ACL insertion, in which dense collagen fibers were connected to the bone by the fibrocartilaginous layer, was microscopically identified at the region between the posteromedial articular cartilage margin of the lateral femoral condyle and the linear bony ridge 7 to 10 mm anterior to the articular cartilage margin. Meticulous comparison of histologic analysis and the 3D VR CT model showed that the ACL direct insertion coincided with a crescent-shaped hollow just behind the linear bony ridge. The direct insertion measured 17.4 +/- 0.9 mm (mean +/- SD) in length, 8.0 +/- 0.5 mm in width, and 128.3 +/- 10.5 mm(2) in area. CONCLUSIONS: The direct insertion of the ACL is located in the depression between the resident's ridge and the articular cartilage margin on the lateral femoral condyle. It measured 17.4 +/- 0.9 mm in length, 8.0 +/- 0.5 mm in width, and 128.3 +/- 10.5 mm(2) in area. CLINICAL RELEVANCE: Delineation of the ACL femoral direct insertion by 3D VR CT could be a useful tool for planning of accurate femoral tunnel positioning in anatomic ACL reconstruction.

Modulation of gene expression and collagen production of anterior cruciate ligament cells through cell shape changes on polycaprolactone/chitosan blends.

Our previous study has illustrated that chitosan could enhance human anterior cruciate ligament (ACL) cells to exhibit a dramatic effect on increasing the gene expression of transforming growth factor beta1 (TGF-beta1), which is a specific gene for wound healing and collagen synthesis. However, human ACL cells could not adhere and proliferate well on chitosan. In order to overcome this drawback, we introduced polycaprolactone (PCL) into chitosan by the method of blending in this study. It was found that the morphology, viability and gene expression of human ACL cells on the chitosan/PCL blends could be effectively regulated. With the increase of PCL content in blends, human ACL cells presented more flatten shape, well-organized cytoskeleton, and higher proliferated ability. Compared to flatten shape, human ACL cells with round shape exhibited higher levels of mRNA expression of TGF-beta1 and collagen type III through 3-day culture period. Furthermore, these blended materials could upregulate protein synthesis of human ACL cells, which corresponded to their gene expressions. Therefore, it is possible to combine the advantages of chitosan and PCL to create a new blended material, which could control cellular morphologies specifically, and further to regulate the gene expression and protein production of cells for specific applications. We expected this concept, controlling the cell shape through biomaterial to modulate the behavior of cells, could provide a new vision for the material selection of ligament tissue engineering.

Platelets and plasma proteins are both required to stimulate collagen gene expression by anterior cruciate ligament cells in three-dimensional culture.

Collagen-platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis.