Effects of low-dose X-ray medical diagnostics on female gonads: Insights from large animal oocytes and human ovaries as complementary models.

Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21-25 and 33-36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33-36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age.

Description of the Follicular Fluid Cytokine and Hormone Profiles in Human Physiological Natural Cycles.

PURPOSE: Exogenous gonadotrophins administration during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles could significantly alter the endogenous follicular regulation system and could influence oocyte quality. The analysis of the follicular fluid (FF) cytokine and hormone profiles in physiological natural cycles is crucial to appreciate the role of FF milieu on follicle development. So far, the FF cytokine profile has been analyzed only in controlled ovarian stimulation cycles and in modified natural cycles. Our study defines, in physiological natural cycles, the cytokine and hormone profiles of individual FF aspirated from antral follicles. METHODS: A total of 203 FFs obtained from 83 women with regular menstrual cycles undergoing ovarian tissue cryopreservation were analyzed: 115 FFs from Group 1 (10 to 29 years of age) and 88 FFs from Group 2 (30 to 40 years of age). In individual FF, 27 cytokines were measured with xMAP technology, and progesterone, estrone, estradiol, testosterone, androstenedione concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: FF hormone profiles were not different in follicular and luteal phase, suggesting that FF hormones are regulated independently of the endogenous gonadotrophins-possibly because 74% of the punctured follicles, which were ≤6 mm, did not require cyclic pituitary function. The follicle size was influenced not only by the FF cytokine profile but also by the FF hormone profile, both of which are dependent on age. MAIN CONCLUSIONS: In physiological natural cycles, FF hormones seems to be regulated independently of the endogenous gonadotropins. Age influences FF hormone and cytokine profiles and the compelling relationship between FF hormones and FF cytokines could influence the follicle development.

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Update on oogenesis in vitro.

INTRODUCTION: Ovarian tissue is increasingly being collected from cancer patients and cryopreserved for fertility preservation. Alternately to the autologous transplantation, the development of culture systems that support oocyte development from the primordial follicle stage represent a valid strategy to restore fertility. The aim of this study is to review the most recent data regarding oogenesis in vitro and to provide an up-to-date on the contemporary knowledge of follicle growth and development in vitro. EVIDENCE ACQUISITION: A comprehensive systematic MEDLINE search was performed since February 2018 for English-language reports by using the following terms: "ovary," "animal and human follicle," "in vitro growth and development," "ovarian tissue culture," "fertility preservation," "IVM," "oocyte." Previous published reviews and recent published original articles were preferred in order to meet our study scope. EVIDENCE SYNTHESIS: Over time, many studies have been conducted with the aim to optimize the characteristics of ovarian tissue culture systems and to better support the three main phases: 1) activation of primordial follicles; 2) isolation and culture of growing preantral follicles; 3) removal from the follicle environment and maturation of oocyte cumulus complexes. While complete oocyte in vitro development has been achieved in mouse, with the production of live offspring, the goal of obtaining oocytes of sufficient quality to support embryo development has not been completely reached into higher mammals despite decades of effort. CONCLUSIONS: Over the years, many improvements have been made on ovarian tissue cultures with the future purpose that patients will be provided with a greater number of developmentally competent oocytes for fertility preservation.

Transplantation of cryopreserved ovarian tissue in a patient affected by metastatic struma ovarii and endometriosis.

In this case report, the outcomes of cryopreserved ovarian tissue transplantation performed in a patient affected by struma-ovarii associated with mature cystic teratoma, recurrent endometriotic cysts and diffuse peritoneal malignant struma-ovarii implants were described. Before cryopreservation, the patient underwent two left ovarian surgeries for enucleation cysts 8 years after righ salpingo-oophorectomy for struma-ovarii. Ovarian biopsy was collected in another hospital and transported to our laboratory for cryopreservation. The patient was submitted to radioiodine-therapy for metastases from malignant struma-ovarii. After treatment she experienced premature ovarian failure. Ten years after cryopreservation, a first orthotopic transplantation was performed in the left ovary and in a peritoneal pocket. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Three years later, a second transplantation was heterotopically performed in abdominal subcutaneous sites. The analysis on thawed ovarian tissue did not reveal micrometastasis and they showed follicle and stroma damages. After transplantation few small follicles were observed at ultrasound examination and hormonal levels remained at menopausal values. To date no ovarian function recovery has been observed. The report highlights that ovarian tissue cryopreservation after multiple ovarian surgery may have some limitations. An accurate counseling should be offered to patients who wish to preserve fertility.

First Italian birth after cryopreserved ovarian tissue transplantation in a patient affected by non-Hodgkin's lymphoma.

This case report describes the first Italian live birth obtained by cryopreserved ovarian tissue transplantation in a woman affected by non-Hodgkin's lymphoma. Before anticancer treatments, several fertility preservation options were proposed. At 29 years the patient underwent laparoscopy for ovarian tissue cryopreservation. After treatments she experienced premature ovarian failure (POF) and asked for cryopreserved ovarian tissue transplantation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Two subsequent ovarian tissue transplantations were performed 4 and 7 years after cryopreservation. The follicle-stimulating hormone and luteinizing hormone reduction, estradiol increase and first menstrual cycle appeared 2 months after the second transplantation. The woman conceived spontaneously 5 months after the second transplantation. After 39 weeks of uneventful gestation, a healthy male baby was born. Ovarian tissue cryopreservation, thawing and transplantation successfully restored ovarian function and fertility after tissue storage.

Long-term storage does not impact the quality of cryopreserved human ovarian tissue.

BACKGROUND: Ovarian tissue cryopreservation is an emerging technique, also addressed to very young cancer patients, for whom it is not possible to perform an ovarian stimulation for oocytes freezing, before gonadotoxic treatment. In this cases, ovarian tissue must be cryopreserved for a long period of time and it is very important to know if it maintains fertility function after a long period of storage. Here we aimed to assess the effect of long-term storage on preservation and viability of cryopreserved human ovarian tissue. METHODS: Descriptive study of three cases of cancer patients whose cryopreserved ovarian tissue remained stored for 18 years. Long-term stored tissue was examined by histological and immunohistochemical analysis, transmission electron microscopy, TUNEL assay and LIVE/DEAD viability/citotoxicity test. RESULTS: Ovarian tissue stored for 18 years showed a good morphology. Follicles presented negative staining for estrogen and progesterone receptors, positive staining for ki67 in granulosa cells and/or oocytes and for bcl2 in granulosa cells. Regarding stroma, patch/focal positive expression was found for estrogen receptor and ki67, diffusely positive expression for progesterone receptor and bcl2. After long-term storage, ultrastructural examination showed sub-cellular integrity of follicles and interstitial oedema foci. No apoptosis was observable by TUNEL assay. Stromal cell viability remained >97 % during the culture period. CONCLUSION: The evaluation of different aspects of the tissue provides evidence that the storage time does not impact on tissue quality and gives hope especially to cancer girls, whose tissues could remain cryopreserved for a very long time.

High cytokine expression and reduced ovarian reserve in patients with Hodgkin lymphoma or non-Hodgkin lymphoma.

OBJECTIVE: To investigate the ovarian reserve in female lymphoma patients and the potential relationships with the cytokine network. DESIGN: Age-matched control study. SETTING: Women's university hospital. PATIENT(S): Seventy-three lymphoma patients (57 with classic Hodgkin lymphoma [HL] and 16 with non-Hodgkin lymphoma [NHL]), approaching our center for ovarian tissue cryopreservation (study group) were compared with 25 age-matched healthy volunteers (control group). INTERVENTION(S): Measurements of antimüllerian hormone (AMH), soluble interleukin-2 receptor (SIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) levels. MAIN OUTCOME MEASURE(S): The AMH and cytokine levels of the lymphoma patients and the healthy volunteers were compared. Correlations between AMH with SIL-2R, IL-6, and IL-8 levels were performed. RESULT(S): The AMH showed significant lower concentrations in lymphoma patients than in the control group. Higher significant concentrations in lymphoma patients than in control group were found for SIL-2R and IL-6. No differences were observed comparing HL and NHL groups and within the stages of HL group for AMH and all the cytokines analyzed. Finally, significant inverse correlations were observed in lymphoma patients between AMH and SIL-2R, IL-6, and IL-8 levels, but not with TNF-α levels. Positive correlations between SIL-2R with IL-6, and IL-6 with IL-8 were also shown. CONCLUSION(S): In patients with HL or NHL at baseline the cytokine network is particularly active and the ovarian reserve is reduced. A strong negative correlation between AMH and SIL-2R, IL-6, and IL-8 has been also evidenced.

Doxorubicin and cisplatin induce apoptosis in ovarian stromal cells obtained from cryopreserved human ovarian tissue.

AIM: To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. PATIENTS & METHODS: Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by 'Live/Dead' and sulforhodamine-B assays, the expression of different apoptosis markers. RESULTS: Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. CONCLUSION: DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.

New insights in the selection and management of cancer patients applicants for ovarian tissue cryopreservation.

Ovarian tissue cryopreservation (OTC), representing a promising strategy to preserve ovarian function in cancer patients, is recommended to women younger than 35 years. This study aimed to identify endocrine and biometric parameters as additional selection criteria for OTC. One hundred and ninety-one cancer patients before chemoradiotherapy and OTC and 43 controls were investigated. Mean ± SD, median, quartiles, 5th and 95th centiles and correlations of FSH, LH, estradiol, inhibin-B, anti-Mullerian hormone (AMH), ovarian volume and antral follicle count (AFC) were assessed. Most ovarian reserve parameters presented typical variations of ovulatory menstrual cycle, except AMH and AFC showing minimal fluctuations across the menstrual cycle. The 5th centiles of AMH (0.31and 0.4 ng/mL in controls and cancer patients, respectively) and AFC (five follicular structures in both groups) could be conjectured as minimum thresholds to include patients aged <35 years in OTC; below this threshold patients of any age should be excluded from OTC. Conversely, patients with AMH and AFC above the 25th centiles (1.2-1.6 ng/mL and 9-10 follicular structures in controls and cancer patients, respectively) might be inserted in OTC regardless of age. Baseline assessment of AMH and AFC might be considered as selection criteria, in addition to chronological age, to take decision of OTC in cancer patients.

Effects of N-acetylcysteine on human ovarian tissue preservation undergoing cryopreservation procedure.

The aim of the study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC), added in freezing/thawing solutions, on reactive oxygen species (RRS) levels and on ovarian tissue preservation after cryopreservation. Ovarian samples from 10 subjects suffering from cancer diseases were cryopreserved using the slow freezing/rapid thawing standard protocol without or with NAC supplementation. RRS levels produced during cryopreservation were monitored by electron paramagnetic resonance (EPR) spectroscopy. The preservation of fresh ovarian tissue (t0), thawed tissue (t1 and t1 NAC) and thawed tissue maintained at 4°C for 2 hrs (t2 and t2 NAC) was analysed by light microscopy, transmission electron microscopy, Ki67 immunohistochemical and TUNEL analysis. It was possible to design a maximum peak for RRS production at t1, which slightly decreased at t2. NAC reduced the extent of RRS levels in cryopreserved ovarian tissues if compared with non-supplemented ones, although not restoring RRS production to baseline values. Comparative analysis between the two cryopreservation protocols showed that a better preservation of morphological characteristics, proliferation index and DNA integrity of ovarian tissue was obtained using NAC and no differences between t1NAC and t2NAC were observed. The employment of NAC during cryopreservation procedure could be an useful strategy for preserving the function of endogenous cellular systems. Nevertheless, further studies on the viability of thawed ovarian tissue are needed to support the feasibility of this approach in clinical settings.

Anti-Müllerian hormone as an ovarian reserve marker in young cancer women who undergo ovarian tissue cryopreservation.

AIM: To evaluate if anti-Müllerian hormone (AMH) is a reliable marker of ovarian reserve in young women undergoing ovarian tissue cryopreservation. PATIENTS & METHODS: Relationships of serum AMH levels with primordial follicle density, age and reproductive hormones were investigated using the Pearson or Spearman correlation coefficient in 86 women with cancer (12-38 years) undergoing ovarian tissue cryopreservation. AMH variations through the menstrual cycle were assessed by the Kruskal-Wallis test. p < 0.05 was accepted as significant. RESULTS: AMH positively correlated with primordial follicle density (p = 0.03), showed great interindividual variability at all ages and negatively correlated with estradiol (p = 0.007) in the early follicular phase. AMH did not vary across the menstrual cycle (p = 0.415). CONCLUSION: AMH appears a valid ovarian reserve marker in young cancer women.

Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification.

The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18-38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

A wide range of 3243A>G/tRNALeu(UUR) (MELAS) mutation loads may segregate in offspring through the female germline bottleneck.

Segregation of mutant mtDNA in human tissues and through the germline is debated, with no consensus about the nature and size of the bottleneck hypothesized to explain rapid generational shifts in mutant loads. We investigated two maternal lineages with an apparently different inheritance pattern of the same pathogenic mtDNA 3243A>G/tRNALeu(UUR) (MELAS) mutation. We collected blood cells, muscle biopsies, urinary epithelium and hair follicles from 20 individuals, as well as oocytes and an ovarian biopsy from one female mutation carrier, all belonging to the two maternal lineages to assess mutant mtDNA load, and calculated the theoretical germline bottleneck size (number of segregating units). We also evaluated "mother-to-offspring" segregations from the literature, for which heteroplasmy assessment was available in at least three siblings besides the proband. Our results showed that mutation load was prevalent in skeletal muscle and urinary epithelium, whereas in blood cells there was an inverse correlation with age, as previously reported. The histoenzymatic staining of the ovarian biopsy failed to show any cytochrome-c-oxidase defective oocyte. Analysis of four oocytes and one offspring from the same unaffected mother of the first family showed intermediate heteroplasmic mutant loads (10% to 75%), whereas very skewed loads of mutant mtDNA (0% or 81%) were detected in five offspring of another unaffected mother from the second family. Bottleneck size was 89 segregating units for the first mother and 84 for the second. This was remarkably close to 88, the number of "segregating units" in the "mother-to-offspring" segregations retrieved from literature. In conclusion, a wide range of mutant loads may be found in offspring tissues and oocytes, resulting from a similar theoretical bottleneck size.

Autotransplantation of cryopreserved ovarian tissue in oncological patients: recovery of ovarian function.

AIM: To present preliminary results of autotransplantation of cryopreserved ovarian tissue performed at Sant'Orsola-Malpighi Hospital, Bologna, Italy. MATERIALS & METHODS: Orthotopic transplantation was performed in two women with colorectal and breast cancer, and heterotopic transplantation was performed in one Hodgkin's lymphoma woman. The presence of micrometastasis in the ovarian tissue was checked, and morphological features of ovarian tissue were evaluated before transplantation. Ovarian function was monitored by hormonal and ultrasound-color Doppler examination after transplantation. RESULTS: In all three women, no micrometastasis was found; light and transmission electron microscopy showed well-preserved thawed ovarian tissue. Ovarian function recovery was observed 2-4 months after transplantation. Spontaneous menstrual cycles occurred in two women with normal follicular densities. No periods occurred in the woman with low follicular density at the time of tissue collection. CONCLUSION: Ovarian tissue cryopreservation and transplantation is a promising approach for preserving ovarian function in women with cancer.

Confocal laser scanning microscopy analysis of bioenergetic potential and oxidative stress in fresh and frozen-thawed human ovarian tissue from oncologic patients.

OBJECTIVE: To evaluate the effectiveness of a bioenergy/oxidative stress assessment based on confocal laser scanning microscopy (CLSM) in association with morphology and ultrastructure analyses based on light microscopy (LM) and transmission electron microscopy (TEM), to monitor the preservation status of cryopreserved human ovarian tissue from cancer patients. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Fourteen young cancer patients. INTERVENTION(S): Human ovarian tissue biopsy, slow freezing/rapid thawing, LM, TEM, CLSM assessment of mitochondrial distribution and activity, and intracellular reactive oxygen species (ROS) localization and levels. MAIN OUTCOME MEASURE(S): In tissue examined before and after slow freezing/rapid thawing, follicular and stromal LM-based score of morphologic damage, ultrastructure, mitochondrial distribution pattern, reactive oxygen species (ROS) localization; mean ± standard deviation of stromal mitochondrial activity and ROS levels. RESULT(S): Severe (n = 6 patients), slight (n = 6 patients), or no (n = 2 patients) LM/TEM-based damage was found in fresh tissue. After freezing/thawing, no further morphologic/ultrastructural alterations were found; however, statistically significant reductions, increases, or no changes in mitochondrial activity and ROS levels were found in severely, slightly, and undamaged tissue, respectively. CONCLUSION(S): Bioenergy/oxidative functional damage was found in tissue with severe LM/TEM-assessed damage. In tissue with slight LM/TEM-assessed damage, the CLSM-based bioenergy/oxidative stress assessment was the only test that allowed discrimination between tissue that had been better (low/no difference) or worse preserved (significant differences).

Cryopreservation of ovarian tissue in breast cancer patients: 10 years of experience.

AIM: To present a decade of experience with ovarian tissue cryopreservation in breast cancer patients. MATERIALS & METHODS: The safety of the procedure was histologically evaluated before and after freezing in 94 patients. Out of 94 patients, 48 prechemotherapy patients were randomly selected to determine stroma and follicle preservation and follicular density. RESULTS: The ovarian tissue from 94 patients did not identify any micrometastases. After cryopreservation, morphology of the ovarian tissue and density of healthy follicles were similar in fresh and frozen tissue. Follicular density decreased with the increasing age of patients in both fresh and frozen tissue (p < 0.0001). A variation in follicular density was observed between fresh and frozen tissue (p < 0.05). CONCLUSION: These results suggest that ovarian tissue cryopreservation is highly feasible for preserving the fertility of young breast cancer patients.

Effects of cyclic increase in gonadotropins on the in vitro development of primordial follicles to antral stage.

The aim of this study was to evaluate the effects of FSH and LH on follicle development during a long-term culture of cryopreserved human ovarian tissue, using morphological and ultrastructural examinations. Thawed ovarian tissue slices from a 4-year-old child with Wilms tumor were cultured for 32 weeks in two different culture conditions, without (medium A) and with (medium B) a monthly peaked increase in FSH and LH. At week 32, in the medium B cultured tissue, a cluster of preantral follicles associated with two oocytes prematurely ovulated was observed, suggesting that the cyclic increase of gonadotropins promoted thawed follicles to grow up to the antral stage. However, the integrity and coordinated follicle development were not maintained. Indeed, ultrastructural analysis showed a well-preserved "naked" oocyte with concomitant features of immaturity and maturity, as if this culture condition had led to an asynchronous maturation of oocyte cytoplasmic components.

Contribution of glycosaminoglycans to the microstructural integrity of fibrillar and fiber crimps in tendons and ligaments.

The biomechanical roles of both tendons and ligaments are fulfilled by the extracellular matrix of these tissues. In particular, tension is mainly transmitted and resisted by protein (collagen, elastin) fibers, whereas compression is opposed by water-soluble glycosaminoglycans (GAGs). GAGs spanning the interfibrillar spaces and interacting with fibrils through the interfibrillar proteoglycans also seem to play a part in transmitting and resisting tensile stresses. Both tendons and ligaments showing similar composition, but different functional roles and collagen array, exhibit periodic undulations of collagen fibers or crimps. Each crimp is composed of many knots of each single fibril or fibrillar crimps. Fibrillar and fiber crimps play a mechanical role in absorbing the initial loading during elongation of both tendons and ligaments, and in recoiling fibrils and fibers when tissues have to return to their original length. This study investigated whether GAGs covalently attached to proteoglycan core proteins directly affect the 3D microstructural integrity of fibrillar crimp regions and fiber crimps in both tendons and ligaments. Achilles tendons and medial collateral ligaments of the knee from eight female Sprague-Dawley rats (90 days old) incubated in a chondroitinase ABC solution to remove GAGs were observed under a scanning electron microscope (SEM). In addition, isolated fibrils of these tissues obtained by mechanical disruption were analyzed by a transmission electron microscope (TEM). Both Achilles tendons and medial collateral ligaments of the rats after chemical or mechanical removal of GAGs still showed crimps and fibrillar crimps comparable to tissues with a normal GAG content. All fibrils in the fibrillar crimp region always twisted leftwards, thus changing their running plane, and then sharply bent, changing their course on a new plane. These data suggest that GAGs do not affect structural integrity or fibrillar crimp functions that seem mainly related to the local fibril leftward twisting and the alternating handedness of collagen from a molecular to a supramolecular level.

Collagen fibre arrangement and functional crimping pattern of the medial collateral ligament in the rat knee.

Ligaments have been described as multifascicular structures with collagen fibres cross-connecting to each other or running straight and parallel also showing a waviness or crimping pattern playing as a shock absorber/recoiling system during joint motions. A particular collagen array and crimping pattern in different ligaments may reflect different biomechanical roles and properties. The aim of the study was to relate the 3D collagen arrangement in the crimping pattern of the medial collateral ligament (MCL) to its functional role. The MCL is one of the most injured ligaments during sports activities and an experimental model to understand the rate, quality and composition of ligaments healing. A deep knowledge of structure-function relationship of collagen fibres array will improve the development of rehabilitation protocols and more appropriate exercises for recovery of functional activity. The rat MCL was analysed by polarized light microscopy, confocal laser microscopy and scanning electron microscopy (SEM). Histomorphometric analysis demonstrated that MCL crimps have a smaller base length versus other tendons. SEM observations demonstrated that collagen fibres showing few crimps were composed of fibrils intertwining and crossing one another in the outer region. Confocal laser analyses excluded a helical array of collagen fibres. By contrast, in the core portion, densely packed straight collagen fibres ran parallel to the main axis of the ligament being interrupted both by planar crimps, similar to tendon crimps, and by newly described right-handed twisted crimps. It is concluded that planar crimps could oppose or respond exclusively to tensional forces parallel to the main ligament axis, whereas the right-handed twisted crimps could better resist/respond to a complex of tensional/rotational forces within the ligament thus opposing to an external rotation of tibia.