Fresh osteochondral grafting in the United States: the current status of tissue banking processing Abstract The use of musculoskeletal allografts has become increasingly popular among surgeons. The purpose of this review is to highlight the procurment and delievery process of fresh osteochondral allografts in the United States. The four distributors of fresh osteochondral allografts in the United States were contacted. Surveys containing quantitative and qualitative sections concerning the procurement and processing of osteochondral allograft tissue were obtained. Our results showed an average of 13 ± 4.24 years of experience with osteochondral allografts. The average donor age ranged from 13.5 ± 3 to 37.5 ± 5 years, with an average age of 27 ± 2.83 years. All donors were between ages 12 and 45 years old. The percentage of screened donors that were accepted for allograft transplant was consistent at 70–75% for 3 out of the 4 tissue banks. The percentage of grafts that expire without implantation ranged from 20% to 29%. Maximum shipping time varied between 24 and 96 hours. Each tissue bank used its own proprietary storage medium. The time from donor death to the harvest of allograft tissue was < 24 hours. The most commonly requested osteochondral allograft tissue for all banks was the medial femoral condyle. The market share of fresh allografts is as follows: Joint Restoration Foundation (JRF) 59.9%, Muskuloskeletal Transplant Foundation (MTF) 15.3%, LifeNet Health (LN) 14.5%, and Regeneration Technology Incorporated (RTI) 10.2%, with approximately 4700 fresh allografts distributed in 2018. This compiled data from the four tissue banks that supply fresh osteochondral allograft in the United States  provides important background information for patients and orthopaedic surgeons.

Correlation between sperm motility and sperm chromatin/DNA damage before and after cryopreservation and the effect of folic acid and nicotinic acid on post-thaw sperm quality in normozoospermic men Abstract Cryopreservation exposes sperm to physical and chemical stresses causing cell damages and impairs sperm functions. The aim of this study was to evaluate the association between motility and sperm chromatin/DNA damage before and after cryopreservation and investigate the effects of folic acid and nicotinic acid on post-thaw sperm quality. Thirty semen samples were obtained from 30 normozoospermic men, aged between 25 and 45 years old. Each sample were divided into five aliquots to form the following groups: fresh, cryopreserved with sperm-freeze only (control), with nicotinic acid (10 mM), with folic acid (50 nM), and with a combination of folic acid (50 nM) + nicotinic acid (10 mM). Sperm viability and motility in each group were assessed by eosin-nigrosine staining and computer-aided sperm analysis respectively. Sperm chromatin quality was studied by aniline blue, toluidine blue, acridine orange staining methods and sperm chromatin dispersion test. Cryopreservation led to a significant reduction in sperm quality in comparison to fresh sample groups (p < 0.05). Sperm chromatin damage was negatively correlated with the percentage of progressively motile cells. Supplementation of the cryopreservation medium with folic acid or nicotinic acid induced a significant improvement in sperm parameters and chromatin quality, compared to control groups (p < 0.05). Meanwhile, the combination of folic acid + nicotinic acid showed a significant protective effect in post thaw sperm. In conclusion, cryopreservation generated oxidative stress, inducingsperm cryodamage, reducing progressive motility and sperm quality, as an indicator of significant chromatin/DNA damage. Folic acid and nicotinic acid exhibited a potential cryoprotective effect by enhancing sperm quality.

In vitro osteodifferentiation of intact human amniotic membrane is not beneficial in the context of bone repair Abstract The human amniotic membrane (hAM) is an attractive biomaterial for regenerative medicine, as it contains amniotic mesenchymal stromal cells (hAMSC), epithelial cells (hAEC) and growth factors. We examined the potential use of hAM in orthopaedic and maxillofacial bone surgery, integrating the requirements of current regulations regarding advanced therapy medicinal products (ATMP) in the European Union. Previous studies have described the potential osteodifferentiation of intact hAM during whole-tissue culture in osteogenic conditions. The present study aims to determine whether in vitro osteodifferentiation of hAM is needed in the context bone repair, and the influence of this process on tissue structure, cell phenotype and cell function. Different conditions (fresh or cultured hAM; intact or hAM-derived cells) were tested. Phenotypic and functional analyses were performed with standard approaches (cell culture and staining, histological and immunolabelling) as well as original approaches (tissue staining, energy dispersive X-ray and X-ray diffraction). In our study, non-osteodifferentiated hAM (i.e., fresh or native hAM) exhibited innate pre-osteoblastic potential. Osteodifferentiation of fresh hAM induced a change in tissue structure, cell phenotype and function. Therefore, we hypothesize that pre-osteodifferentiation may not be necessary, especially if it induces unwanted changes. To our surprise, in these osteogenic conditions, hAEC had a mesenchymal phenotype with osteocyte function, and even native synthesis of hydroxyapatite, focusing osteogenic potential mainly in this epithelial layer. In conclusion, in vitro osteodifferentiation by tissue culture does not appear to be necessary for hAM to be used as an innovative ATMP for bone repair.

Effect of different cryopreservation regimens on Ehrlich carcinoma growth Abstract The freezing rate is a decisive factor in determining the purpose of using low temperatures, i.e., for cryoablation or cryobanking of tumor cells. The research aim was to determine effect of different cryopreservation regimens on Ehrlich carcinoma (EC) growth in vivo and subpopulation composition of the formed ascites. The previously cryopreserved with slow and rapid rates EC cells were cultured in peritoneal cavity (PC) of mice for 7 days. Absolute number of cells in the PC, the subpopulation composition of tumor with flow cytometry using CD44 and CD24 markers were determined. Immediately after warming, a significant redistribution of EC subpopulation composition with a decreased content of the most tumorigenic CD44high cells after both freezing regimens was found. Culturing in vivo for 7 days contributed to the restoration of EC subpopulation composition, but with some a decrease in the tumor growth intensity when slow cooling was used. Rapid cooling contributed to significant inhibition of tumor growth with a reduced number of CD44+ and increased CD24+ cells. None of the cryopreservation regimens resulted in a complete elimination of tumorigenic CD44high tumor cells. The freezing rate determines the preservation of the subpopulation composition of the EC and intensity of its growth in vivo.

Microbiological contamination in donor corneas preserved for medium-term Abstract To evaluate the characteristics of microbiological contamination in donor corneas preserved for medium-term. A total of 82 donated corneas from June 1, 2014 to November 30, 2014 were retrospectively analyzed. The corneas were preserved in cornea chambers medium-term solution at 4–8 °C for keratoplasty. After removal of the central corneas for transplantation, the corneoscleral rims were put back into the medium for 1 month at room temperature (20–25 °C). The suspicious contaminated storage solutions indicated with transparency or color change were examined with bacteria and fungi cultivation for strain identification. The data collected included gender, age, procurement site and causes of death of donors, and follow-up of recipients. Statistical analysis was performed using Microsoft Excel and SPSS 24.0. Significance level was set at a P value < 0.05. The overall pathogen positive rate was 9.8% (n = 8), including 7 (87.5%) fungi and 1 (12.5%) bacteria. They were 2 (2.44%) Fusarium, 2 (2.44%) Chromomycosis, 1 (1.22%) Candida albicans, 1 (1.22%) Aspergillus versicolor, 1 (1.22%) Acremonium species, and 1 (1.22%) Enterococcus. 5 contaminated corneas were used for penetrating keratoplasty; although four out of five (80%) had not been given antifungal drugs during more than 6 months following-up period, none of the recipients was infected with a graft. Donor age (P = 0.839), gender (P = 0.062), procurement sites (P = 0.713) and cause of death (P = 0.711) had no statistically significant influence on the contamination rate. All donor corneas have a possibility of microbiological contamination. Strict tissue preservation protocol but not antifungal drugs following keratoplasty seems necessary to prevent graft infection.

Does supplementation of sperm freezing/thawing media with Ceratonia siliqua improve detrimental effect of cryopreservation on sperm parameters and chromatin quality in normozoospermic specimens? Abstract Human sperm banking is an important procedure in the assisted reproductive technique centers. It entails sperm damage. The aim of this study was to investigate beneficial effect of Ceratonia siliqua (C. siliqua) supplement in freezing/thawing media on post thaw sperm parameters and sperm chromatin quality in normozoospermic samples. Forty normozoospermic specimens were included in this prospective study. Each sample was divided into ten groups. In groups one to five, 0 (as control group) 5, 10, 20 and 30 µg/ml C. siliqua were added to freezing medium and in groups six to ten, similar concentration of C. siliqua were added to thawing medium for 30 min incubation. Sperm concentration, progressive motility, normal morphology, viability, aniline blue (AB), toluidine blue (TB) and sperm chromatin dispersion (SCD) staining tests were evaluated before vitrification and after thawing. The results showed that 10 and 20 µg/ml supplementation of C. siliqua in freezing/thawing media significantly increased progressive motility, normal morphology and viability of sperm (p < 0.05) as well as decreased AB, TB and SCD (p < 0.05). Also, 20 µg/ml had significantly higher improvement compared to 10 µg/ml C. siliqua (p < 0.05). The present study showed that C. siliquasupplemented freezing/thawing media can improve sperm quality of normozoospermic samples after freezing/thawing.

Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro Abstract The extracellular matrix (ECM) is a dynamic and intricate three-dimensional (3D) microenvironment with excellent biophysical, biomechanical, and biochemical properties that may directly or indirectly regulate cell behavior, including proliferation, adhesion, migration, and differentiation. Compared with tissue-derived ECM, cell-derived ECM potentially has more advantages, including less potential for pathogen transfer, fewer inflammatory or anti-host immune responses, and a closer resemblance to the native ECM microenvironment. Different types of cell-derived ECM, such as adipose stem cells, synovium-derived stem cells and bone marrow stromal cells, their effects on articular chondrocytes which have been researched. In this study, we aimed to develop a 3D cell culture substrate using decellularized ECM derived from human umbilical cord-derived mesenchymal stem cells (hUCMSCs), and evaluated the effects on articular chondrocytes. We evaluated the morphology and components of hUCMSC-derived ECM using physical and chemical methods. Morphological, histological, immunohistochemical, biochemical, and real-time PCR analyses demonstrated that proliferation and differentiation capacity of chondrocytes using the 3D hUCMSC-derived ECM culture substrate was superior to that using non-coated two-dimensional plastic culture plates. In conclusion, 3D decellularized ECM derived from hUCMSCs offers a tissue-specific microenvironment for in vitro culture of chondrocytes, which not only markedly promoted chondrocyte proliferation but also preserved the differentiation capacity of chondrocytes. Therefore, our findings suggest that a 3D cell-derived ECM microenvironment represents a promising prospect for autologous chondrocyte-based cartilage tissue engineering and regeneration. The hUCMSC-derived ECM as a biomaterial is used for the preparation of scaffold or hybrid scaffold products which need to further study in the future.

An anticancer effect of umbilical cord-derived mesenchymal stem cell secretome on the breast cancer cell line Abstract Nowadays, Mesenchymal stem cells (MSCs) have become one of the most attractive tools for treating tumors, due to their specific characteristics, the most prominent of which are tropism toward tumor. These cells will exert their effects through their secretion. In this study, our aim was to evaluate the anti-cancer effect of umbilical cord-derived mesenchymal cells (UCMSC) secretome, on MCF-7 tumor cells. MSCs were extracted from the umbilical cord of mothers, having normal delivery or cesarean section. After culture, the supernatants of these cells were collected and freeze-dried. The cytotoxic effect of freeze-dried secretome was examined at different concentrations on MCF-7 and the optimum concentrations (IC50) were calculated, using MTT assay. These results were confirmed by BrdU assay. The effect of induction of apoptosis of the MSC secretome on MCF-7 was determined, using annexin V/PI method by flow cytometry. The results of our study indicate that the isolation and growth time of UCMSCs of mothers who were naturally delivered was lower than those who received cesarean section. Co-culture studies showed that MSCs had cytotoxic effects on MCF-7 cells. The MSC secretome also showed cytotoxic effects on the MCF-7 cell line, this effect was mediated by induction of apoptosis, which was dose-dependent with an IC50 of 10 mg/mL.

Decellularized and solubilized pancreatic stroma promotes the in vitro proliferation, migration and differentiation of BMSCs into IPCs Abstract Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into insulin-producing cells (IPCs). Bio-scaffolds derived from decellularized organs can act as a carrier for seed cells and may have broad applications in regenerative medicine. This study investigated the effect of native pancreatic stroma obtained from decellularized pancreas on the proliferation, migration and differentiation of BMSCs into IPCs, and explored the potential underlying molecular mechanism. The decellularized pancreas bio-scaffold was obtained by perfusion with Triton X-100/ammonium hydroxide, followed by digestion with a mixture of pepsin and hydrochloric acid to prepare the stroma solution. Islet-like cells were differentiated from BMSCs by a three-step induction method. The differences on the cytological behavior with or without stroma were evaluated by morphological observation, insulin release assay, qRT-PCR assay and western blot analysis. Our results showed that, stroma derived from decellularized pancreas could promote the proliferation and migration of BMSCs. Furthermore, the formation of IPCs could also be promoted, which possessed similar morphology to endogenous islets. During the induced differentiation process, the presence of stroma significantly increased the expression of insulin 1, insulin 2 and Pdx-1, as well as insulin release. This was accompanied by an increase in the phosphorylation of Akt and ERK in third stage cell clusters, which was prevented by the addition of the inhibitors PD98059 and LY294002, respectively. In summary, decellularized pancreatic stroma could promote the proliferation, migration and differentiation of BMSCs into IPCs, and this involved the activation of Akt and ERK signal pathways.

Evaluating the adhesion of human gingival fibroblasts and MG-63 osteoblast-like cells to activated PRP-coated membranes Abstract Regeneration of periodontal tissues is affected by the biological and morphological characteristics of the membrane surface. The current study evaluated the adhesion of human gingival fibroblasts (HGF) and MG-63 osteoblast-like cells to Membranes, with and without activated PRP. The line of human gingival fibroblast cells and MG-63 osteoblast-like cells were first prepared and cultured on three types of membranes, including Jason, CenoMembrane and TXT-200 in three groups (FBS 10%, FBS 0.5% and activated PRP). Cell viability was investigated by MTT assay and electron microscopy (SEM) was used to evaluate the cell morphology and adhesion on these membranes after 24 and 72 h. Two-way ANOVA was carried out at the significance level of 0.05. The highest adhesion in the 10% FBS group for HGF and The MG-63 osteoblast-like cells was observed to the Jason membrane during 24 h and 72 h (p < 0.05). However, there were no significant differences among the three membranes in PRP and FBS groups for HGF during 24 h and for MG-63 cells during 72 h (p > 0.05). Activated PRP had a positive effect on the viability and adhesion of both human gingival fibroblasts and osteoblast-like cells as compared to the FBS 0.5% group, but these effects were not as 10% FBS group. The results also showed that Jason membrane had the highest amount of cell viability and adhesion.