The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon

The purpose of the present study is to determine the effect of the stress enhancement and intrinsic fibroblasts on the extracellular matrix of the patellar tendon. Thirty-two female Japanese White rabbits were divided into four groups. In Group 1, the patellar tendon underwent the in situ freeze-thaw treatment to kill intrinsic fibroblasts of the patellar tendon and the patellar tendon underwent the wrapping treatment with nylon membrane filters to inhibit extrinsic cell infiltration. In Group 2, the medial and the lateral portions of the frozen-thawed patellar tendon were resected to enhance the stress, and then the central two-thirds of the patellar tendon underwent the wrapping treatment. In Group 3, the patellar tendon without the freeze/thaw treatment underwent the wrapping treatment. In Group 4, the patellar tendon was narrowed and wrapped in the same manner. All rabbits were killed 6 weeks after surgery. While the elastic modulus and the tensile strength of the patellar tendon in Group 2 were significantly less than those in Group 1, we could not find any significant differences in these parameters between Groups 3 and 4. Histologically, while no fibroblasts were observed in Groups 1 and 2, fibroblasts were found in Groups 3 and 4. This study revealed that stress enhancement decreases the elastic modulus and the tensile strength of the extracellular matrix of the patellar tendon and that intrinsic fibroblasts prevent the detrimental effect of stress enhancement on mechanical properties of the patellar tendon.     

Antibody neutralization of TGF-beta enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration

A tissue-cultured tendon matrix infiltrated with cultured fibroblasts can be regarded as an ideal tissue-engineered tendon model. To clarify the role of TGF-beta in a tissue-cultured tendon matrix during ex vivo cellular infiltration, the present ex vivo study was conducted to test the following hypothesis that antibody neutralization of TGF-beta enhances weakening of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. In skeletally mature female rabbits, fibroblasts were isolated from the right patellar tendons using an explant culture technique, and the left patellar tendons underwent multiple freeze/thaw treatment with liquid nitrogen to obtain an acellular tendon matrix. Each acellular tendon was placed in a collagen gel containing cultured fibroblasts and then incubated with or without anti-TGF-beta1 antibody for 6 weeks. We found that antibody neutralization of TGF-beta enhanced the decrease in the tensile strength and tensile modulus of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. The present study indicates a possibility that TGF-beta may have a role in suppressing the material deterioration of the fascicles in the tendon during ex vivo cellular infiltration.     

Local administration of interleukin-1 receptor antagonist inhibits deterioration of mechanical properties of the stress-shielded patellar tendon

We previously found that interleukin (IL)-1beta is over-expressed in the fibroblasts of the stress-shielded patellar tendon using a stress-shielding model [Uchida, H., Tohyama, H., Nagashima, K., Ohba, Y., Matsumoto, H., Toyama, Y., Yasuda, K., 2005. Stress deprivation simultaneously induces over-expression of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta in fibroblasts and mechanical deterioration of the tissue in the patellar tendon. Journal of Biomechanics 38(4), 791-798.]. Therefore, IL-1beta may play a role in tendon deterioration in response to stress deprivation. This study was conducted to clarify the effects of local administration of interleukin-1 receptor antagonist (IL-1ra) on the mechanical properties of the stress-shielded patellar tendon as well as the tendon fascicles harvested from it. Twenty-six mature rabbits were equally divided into Groups IL-1ra and PBS after the right patellar tendon underwent the stress-shielding treatment, which completely released the patellar tendon from tension by stretching the flexible wire installed between the patella and the tibial tubercle. In Group IL-1ra, IL-1ra was injected between the patellar tendon and the infra-patellar fat pad. In Group PBS, phosphate-buffered saline was injected in the same manner as IL-1ra. All rabbits were evaluated at 3 weeks after the stress-shielding procedure. The tangent modulus and the tensile strength of the patellar tendons were significantly greater in Group IL-1ra than in Group PBS, while there was no significant difference in the strain at failure between Groups IL-1ra and PBS. Concerning the mechanical properties of the fascicles harvested from the patellar tendon, however, we could not detect any significant differences in the tangent modulus, tensile strength, or strain at failure between Groups IL-1ra and PBS. The present study suggested that IL-1 plays an important role in the deterioration of the mechanical properties of the patellar tendon in response to stress shielding and that IL-1 does not affect the fascicles themselves.     

Studies in flexor tendon wound healing: neutralizing antibody to TGF-beta1 increases postoperative range of motion

The postoperative outcome of hand flexor tendon repair remains limited by tendon adhesions that prevent normal range of motion. Recent studies using in situ hybridization techniques have implicated transforming growth factor beta-1 (TGF-beta1) in both intrinsic and extrinsic mechanisms of repair. TGF-beta1 is a growth factor that plays multiple roles in wound healing and has also been implicated in the pathogenesis of excessive scar formation. The purpose of this study was to examine the effect of neutralizing antibody to TGF-beta1 in a rabbit zone II flexor tendon wound-healing model. Twenty-two adult New Zealand White rabbits underwent complete transection of the middle digit flexor digitorum profundus tendon in zone II. The tendons were immediately repaired and received intraoperative infiltration of one of the following substances: (1) control phosphate-buffered saline; (2) 50 microg neutralizing antibody to TGF-beta1; (3) 50 microg each of neutralizing antibody to TGF-beta1 and to TGF-beta2. Eight rabbits that had not been operated on underwent analysis for determination of normal flexion range of motion at their proximal and distal interphalangeal joints, using a 1.2-N axial load applied to the flexor digitorum profundus tendon. All rabbits that had been operated on were placed in casts for 8 weeks to allow maximal tendon adhesion and were then killed to determine their flexion range of motion. Statistical analysis was performed using the Student's unpaired t test. When a 1.2-N load was used on rabbit forepaws that had not been operated on, normal combined flexion range of motion at the proximal and distal interphalangeal joints was 93+/-6 degrees. Previous immobilization in casts did not reduce the range of motion in these forepaws (93+/-4 degrees). In the experimental groups, complete transection and repair of the flexor digitorum profundus tendon with infiltration of control phosphate-buffered saline solution resulted in significantly decreased range of motion between the proximal and distal phalanges [15+/-6 degrees (n = 8)]. However, in the tendon repairs infiltrated with neutralizing antibody to TGF-beta1, flexion range of motion increased to 32+/-9 degrees (n = 7; p = 0.002). Interestingly, a combination of neutralizing antibody to TGF-beta1 and that to TGF-beta2 did not improve postoperative range of motion [18+/-4 degrees (n = 7; p = 0.234)]. These data demonstrate that (1) the rabbit flexor tendon repair model is useful for quantifying tendon scar formation on the basis of degrees of flexion between proximal and distal phalanges; (2) intraoperative infiltration of neutralizing antibody to TGF-beta1 improves flexor tendon excursion; and (3) simultaneous infiltration of neutralizing antibody to TGF-beta2 nullifies this effect. Because TGF-beta1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that intraoperative biochemical modulation of TGF-beta1 levels limits flexor tendon adhesion formation.     

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80–90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young’s were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.     

Effect of implanting a soft tissue autograft in a central-third patellar tendon defect: biomechanical and histological comparisons

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n?=?10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to biomechanical (n?=?7) or histological (n?=?3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p?=?0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model.     

Production of a sterilised decellularised tendon allograft for clinical use

Application of a high-level decontamination or sterilisation procedure and cell removal technique to tendon allograft can reduce the concerns of disease transmission, immune reaction, and may improve remodelling of the graft after implantation. The decellularised matrix can also be used as a matrix for tendon tissue engineering. One such sterilisation factor, Peracetic acid (PAA) has the advantage of not producing harmful reaction residues. The aim of this study was to evaluate the effects of PAA treatment and a cell removal procedure on the production of tendon matrix. Human patellar tendons, thawed from frozen were treated respectively as: Group 1, control with no treatment; Group 2, sterilised with PAA (0.1 % (w/v) PAA for 3 h) Group 3, decellularised (incubation successively in hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, and a nuclease solution); Group 4, decellularised and PAA sterilised. Histological analysis showed that no cells were visible after the decellularisation treatment. The integrity of tendon structure was maintained after decellularisation and PAA sterilisation, however, the collagen waveform was slightly loosened. No contact cytotoxicity was found in any of the groups. Determination of de-natured collagen showed no significant increase when compared with the control. This suggested that the decellularisation and sterilisation processing procedures did not compromise the major properties of the tendon. The sterilised, decellularised tendon could be suitable for clinical use.     

Effects of administration of transforming growth factor (TGF)-beta1 and anti-TGF-beta1 antibody on the mechanical properties of the stress-shielded patellar tendon

Previous studies have shown that, in the stress-shielded patellar tendon, the mechanical properties of the tendon were dramatically reduced and TGF-beta was over-expressed in tendon fibroblasts. In the present study, therefore, we tested two supportive hypotheses using 40 rabbits: One was that an application of TGF-beta1 might significantly increase the tensile strength and the tangent modulus of the stress-shielded patellar tendon. The other one was that an administration of anti-TGF-beta1 antibody might significantly reduce the mechanical properties of the stress-shielded patellar tendon. In the results, an application of 4-ng TGF-beta1 significantly increased the tangent modulus of the stress-shielded patellar tendon at 3 weeks (p = 0.019), compared with the sham treatment. Concerning the tensile strength, the 4-ng TGF-beta1 application increased the average value, but a statistical significance was not reached. An application of 50-microg anti-TGF-beta1 antibody significantly reduced the tangent modulus and the tensile strength of the stress-shielded patellar tendon at 3 weeks (p = 0.0068 and p = 0.0355), compared with the sham treatment. Because the stress-shielding treatment used in this study dramatically reduces the tangent modulus and the tensile strength of the patellar tendon, the present study suggested that an administration of TGF-beta1 weakly but significantly inhibited the reduction of the mechanical properties of the stress-shielded patellar tendon, and that inactivation of TGF-beta1 with its antibody significantly enhanced the reduction of the mechanical properties that occurs in the stress-shielded patellar tendon. These results suggested that TGF-beta1 plays an important role in remodeling of the stress-shielded patellar tendon.     

The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts

Recombinant human (rh) bone morphogenetic protein 12 (BMP12) is proved to induce the formation of tendon and ligament tissues in animal experiments. But the roles of BMP12 on tissue regeneration in human tendons remain unexplored. In the present study, healthy human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, only located on active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of PCNA and procollagen type I also exhibited a similar distribution. It indicates that BMP12 may be involved in matrix remodeling process in adult tissues. In vitro studies showed that rhBMP12 could increase proliferation of tendon fibroblasts and increase the gene expression of procollagen type I and type III, but decrease the gene expression of decorin in tendon fibroblasts culture. Our findings suggest that BMP12 may play a role in early phases of tissue regeneration in tendons.     

Development and evaluation of multiple tendon injury models in the mouse

The mouse has proven to be an advantageous animal model system in basic science research focused on aiding in development and evaluation of potential treatments; however, the small size of mouse tendons makes consistent and reproducible injury models and subsequent biomechanical evaluation challenging for studying tendon healing. In this study, we investigated the feasibility and reproducibility of multiple mouse tendon injury models. Our hypothesis was that incisional (using a blade) and excisional (using a biopsy punch) injuries would result in consistent differences in tendon material properties. At 16 weeks of age, 17 C57BL/6 mice underwent surgery to create defects in the flexor digitorum longus, Achilles, or patellar tendon. Each animal received 1-2 full-thickness, central-width incisional or excisional injuries per limb; at least one tendon per limb remained uninjured. The injuries were distributed such that each tendon type had comparable numbers of uninjured, incisionally injured, and excisionally injured specimens. Three weeks after injury, all animals were euthanized and tendons were harvested for mechanical testing. As hypothesized, differences were detected for all three different tendon types at three weeks post-injury. While all models created injuries that produced predictable outcomes, the patellar tendon model was the most consistent in terms of number and size of significant differences in injured tendons compared to native properties, as well as in the overall variance in the data. This finding provides support for its use in fundamental tendon healing studies; however, future work may use any of these models, based on their appropriateness for the specific question under study.     

Shear Elastic Modulus on Patellar Tendon Captured from Supersonic Shear Imaging: Correlation with Tangent Traction Modulus Computed from Material Testing System and Test–Retest Reliability

Characterization of the elastic properties of a tendon could enhance the diagnosis and treatment of tendon injuries. The purpose of this study was to examine the correlation between the shear elastic modulus on the patellar tendon captured from a Supersonic Shear Imaging (SSI) and the tangent traction modulus computed from a Material testing system (MTS) on 8 fresh patellar pig tendons (Experiment I). Test–retest reliability of the shear elastic modulus captured from the SSI was established in Experiment II on 22 patellar tendons of 11 healthy human subjects using the SSI. Spearman Correlation coefficients for the shear elastic modulus and tangent traction modulus ranged from 0.82 to 1.00 (all p<0.05) on the 8 tendons. The intra and inter-operator reliabilities were 0.98 (95% CI: 0.93–0.99) and 0.97 (95% CI: 0.93–0.98) respectively. The results from this study demonstrate that the shear elastic modulus of the patellar tendon measured by the SSI is related to the tangent traction modulus quantified by the MTS. The SSI shows good intra and inter-operator repeatability. Therefore, the present study shows that SSI can be used to assess elastic properties of a tendon.     

Biomechanics of tendon injury and repair

Many clinical and experimental studies have investigated how tendons repair in response to an injury. This body of work has led to a greater understanding of tendon healing mechanisms and subsequently to an improvement in their treatment. In this review paper, characterization of normal and healing tendons is first covered. In addition, the debate between intrinsic and extrinsic healing is examined, and the cellular and extracellular matrix response following a tendon injury is detailed. Next, clinical and experimental injury and repair methods utilizing animal models are discussed. Animal models have been utilized to study the effect of various activity levels, motions, injury methods, and injury locations on tendon injury and repair. Finally, current and future treatment modalities for improving tendon healing, such as tissue engineering, cell therapy, and gene therapy, are reviewed.     

Extensive expression of markers for acetylcholine synthesis and of M2 receptors in tenocytes in therapy-resistant chronic painful patellar tendon tendinosis - a pilot study

We have recently obtained evidence favoring the occurrence of an up-regulation of a non-neuronal cholinergic system in chronic painful patellar tendon tendinosis. It seems possible that this up-regulation to a certain degree may be involved in the manifestations of the disease. Today, there is a new, very successful, line of treatment of patellar tendinosis in the form of Doppler guided sclerosing injections. However, a few patients seem resistant to this therapy. Therefore, we have in this pilot study investigated biopsies from the patellar tendon of three such therapy-resistant patients, using immunohistochemistry. In situ hybridization was also applied. Comparisons were made with a material of specimens from both normal (n=16) and tendinosis (n=7) tendons, also previously examined. The study showed that there were extensive immunoreactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter, as well as for the M(2) muscarinic acetylcholine receptor, in the overwhelming majority of the tenocytes. The immunoreactions were more pronounced than those generally obtained in the tendinosis tissue of the previously studied patients and clearly more pronounced than those of patellar tendon tissue of controls. Also, for the first time, we here present findings of mRNA for ChAT within tenocytes. In conclusion, it appears as if there is an excessive local acetylcholine (ACh) production and an occurrence of marked ACh effects in cases of severe tendinosis. An excessive production of local ACh might be related to pain sensation and the processes that occur in tendinosis development, such as cell proliferation. Thus, the results of this pilot study suggest that non-neuronal ACh is highly involved in the pathology of therapy-resistant patellar tendinosis.     

TGF-β1, TGF-β3, and PGE<Subscript>2</Subscript> regulate contraction of human patellar tendon fibroblasts

To understand the role of tendon fibroblast contraction in tendon healing, we investigated the contraction of human patellar tendon fibroblasts (HPTFs) and its regulation by transforming growth factor-beta1 (TGF-beta1), TGF-beta3, and prostaglandin E(2) (PGE(2)). HPTFs were found to wrinkle the underlying thin silicone membranes, demonstrating that these tendon fibroblasts are contractile. Using fibroblast populated collagen gels (FPCGs), exogenous addition of TGF-beta1 or TGF-beta3 was found to increase fibroblast contraction compared to non-treated fibroblasts in serum-free medium, whereas PGE(2) was found to decrease the tendon fibroblast contraction. Moreover, the tendon fibroblasts in collagen gels treated with TGF-beta1 contracted to a greater degree than those treated with TGF-beta3. Since the extent of fibroblast contraction is related to scar tissue formation, this differential effect of TGF-beta1 and TGF-beta3 on HPTF contraction supports the previous finding that TGF-beta1 induces scar tissue formation, whereas TGF-beta3 reduces its formation. Further, the reduced tendon fibroblast contraction by PGE(2) suggests that excessive presence of this inflammatory mediator in the wound site might retard tendon healing. Taken together, the results of this study suggest that regulation of human tendon fibroblast contraction may reduce scar tissue formation and therefore improve the mechanical properties of healing tendons.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.     

Effect of age on collagen fibril diameter in rabbit patellar tendon repair

The effect of aging on soft tissue repair is poorly understood. We examined collagen fibril diameter in repairing patellar tendons from young adult and aging rabbits. We hypothesized that repairing tendons from older (geriatric) rabbits would have similar diameter fibrils compared with the younger (young adult) rabbits. Full-length, full-thickness, central-third (2.5 to 3 mm) patellar tendon injuries were made by cutting out the center of the tendon in twelve 1-y-old and thirteen 4- to 5.5 (average, 4.25)-y-old female New Zealand White rabbits. The contralateral tendon served as an unoperated control. The rabbits were euthanized at 6, 12, and 26 wk after surgery. The collagen fibril diameter was examined by electron microscopy at the patellar end, middle, and tibial end of the patellar tendon. There was no significant decline in collagen fibril diameter at any location in the aging rabbit healing patellar tendons compared with those of the 1-y-old rabbits. This study found that collagen fibril diameter was not altered with increasing age in the healing rabbit patellar tendon.     

Studying the growth of cervical carcinoma nests and angiogenesis by immunostaining, quantitation and three-dimensional structural analysis

OBJECTIVE: To analyze the relationship and mutual effect of the growth of cervical carcinoma nests and angiogenesis. STUDY DESIGN: Serial paraffin sections of cervical squamous carcinoma were stained in repeated order with hematoxylin-eosin (HE), immunostain for factor VIII-related antigen, type IV collagen and proliferating cell nuclear antigen (PCNA). Three-dimensional reconstructions were made, and the volumes of carcinoma nests, necrosis and microvessels were measured. RESULTS: Two types of cervical carcinoma nests were distinguished on the basis of their growth characteristics and vascularity (groups I and II). Group I nests: The carcinoma cells were proliferating actively, as determined by their morphology and PCNA staining. There were abundant microvessels. Some endothelial sprouts and cords penetrated the nests and then formed networks and new vessels. The volume ratio of microvessels, including sprouts and cords, to the nests was 0.6282:1. Group II nests: The center of these nests underwent necrosis. The peripheral cells were rather small, with no mitosis. The PCNA index was rather low; these nests grew very slowly. There were only a few surrounding microvessels with no endothelial sprouts or cords. The volume ratio of vessels to nest was 0.0657:1. CONCLUSION: Two types of cervical carcinoma nests show a close relationship and mutual effect of the growth of carcinoma nest and angiogenesis. Group I nests grow and develop well, with abundant microvessels. Thus, many tumor cells may be angiogenic and induce angiogenesis; growth of the nests seemed dependent on adequate neovascularization. Group II nests grow slowly, with a few microvessels; the center of the nests undergoes necrosis. The inadequate blood supply must be one of the important causes of necrosis. Considering that there must have been abundant neovascularization during their growth in the past, most of the microvessels must have been obliterated and then reabsorbed to make the remaining vessels so few.     

Re-evaluation of the distinction between type I and type II cells: the necessary role of the mitochondria in both the extrinsic and intrinsic signaling pathways upon Fas receptor activation

Cyclosporin A (CyA) and bongkrekic acid (BK) prevented Fas-induced apoptosis in two type I cell lines (H9 and SKW6.4) and two type II cell lines (Jurkat and CEM). CyA and BK inhibited the release of cytochrome c in all four cell lines. In type I cells and in CEM cells, CyA and BK did not prevent the translocation of Bax to the mitochondria. In these same cells, full-length Bid decreased in the mitochondria and cytosol. The cleavage product of Bid, tBid, appeared in the cytosol and to a lesser extent in the mitochondria. In Jurkat cells, Bid also decreased in the cytosol, but increased in the mitochondria. Similar to the other cells, tBid appeared in the mitochondria and cytosol. In the type I H9 and SKW6.4 cells and type II Jurkat cells, the caspase-8 inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD) prevented the cell killing. In the type I cells, IETD prevented the translocation of Bax, the degradation of Bid and the accumulation of tBid. By contrast, IETD only marginally protected the type II CEM cells. In these cells in the presence of IETD, Bax translocated to the mitochondria, in the absence of any degradation of Bid or accumulation of tBid. In the type I H9 cells, IETD produced a depletion of ATP, an effect that did not occur in the type II CEM cells. It is concluded that in type I cells the extrinsic signaling pathway is mitochondrial dependent to the same extent as is the intrinsic pathway in type II cells.     

Influence of patellar position on the knee extensor mechanism in normal and crouched walking

Patella alta is common in cerebral palsy, especially in patients with crouch gait. Correction of patella alta has been advocated in the treatment of crouch, however the appropriate degree of correction and the implications for knee extensor function remain unclear. Therefore, the goal of this study was to assess the impact of patellar position on quadriceps and patellar tendon forces during normal and crouch gait. To this end, a lower extremity musculoskeletal model with a novel 12 degree of freedom knee joint was used to simulate normal gait in a healthy child, as well as mild (23 deg min knee flexion in stance), moderate (41 deg), and severe (67 deg) crouch gait in three children with cerebral palsy. The simulations revealed that quadriceps and patellar tendon forces increase dramatically with crouch, and are modulated by patellar position. For example with a normal patellar tendon position, peak patellar tendon forces were 0.7 times body weight in normal walking, but reached 2.2, 3.2 and 5.4 times body weight in mild, moderate and severe crouch. Moderate patella alta acted to reduce quadriceps and patellar tendon loads in crouch gait, due to an enhancement of the patellar tendon moment arms with alta in a flexed knee. In contrast, patella baja reduced the patellar tendon moment arm in a flexed knee and thus induced an increase in the patellar tendon loads needed to walk in crouch. Functionally, these results suggest that patella baja could also compromise knee extensor function for other flexed knee activities such as chair rise and stair climbing. The findings are important to consider when using surgical approaches for correcting patella alta in children who exhibit crouch gait patterns.