Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

Different Effects of Sugars and Methods to Preserve Post-Thaw Functional Properties of Cryopreserved Caprine Spermatogonial Stem Cells.

The present study aimed to identify the effects of sugar and methods (slow freezing [SF] vs. fast freezing [FF]) on post-thaw in vitro functional characteristics of cryopreserved caprine spermatogonial stem cells (cSSCs) and the cells obtained from cryopreserved testis tissue of prepubertal Barbari bucks. For this, in experiment 1, cSSCs were isolated and cryopreserved by either SF or FF method with different non-permeable (sugars; trehalose [140 mm; 140T or 400 mm; 400T] and sucrose [140 mm; 140S or 400 mm; 400S]) or/and permeable (5% ethylene glycol [EG] and dimethyl sulfoxide) cryoprotectants. After 1 week of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment 2, the effectiveness of sugars (trehalose [140 mm] or sucrose [140 mm]) for cryopreservation of testicular tissues of prepubertal Barbari bucks using the SF or FF method was evaluated. After 1 week of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3 weeks. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers' expression. The recovery rate was 1.3-, 1.3-, and 1.1-fold higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (protein gene product 9.5 and octamer-binding transcription factor-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue weight was significantly (p < 0.05) higher when cryopreserved using 140 mm trehalose compared with other groups. The results of immunocytochemical analyses imply the expression of pluripotent stem cell markers in cSSCs following cryopreservation. Overall, the outcome of the study demonstrates different effects of sugars and methods on post-thaw functional properties of cSSCs with superiority of 140 mm trehalose using SF method over other treatment groups. These results are important for ex vivo expansion and differentiation of cSSCs for fertility preservation and their other downstream applications.

Successful in vivo Transplantation of Cultured and Enriched Testicular Germ Cells of Pre-Pubertal Bucks to Busulfan-Treated Homologous Recipients.

The objective of the present study was to establish a workable approach for the production of germ cell (GC)-depleted recipient goat model using intra-testicular busulfan treatment and transplantation of cultured and enriched caprine-male GC (cmGCs) into the homologous recipients under ultrasonography (USG) guidance. The evaluation of post-transplantation colonization of donor cmGCs and restoration of the normal architecture of seminiferous tubules (ST) was performed. For this, the cmGCs of pre-pubertal male goats were isolated and enriched by differential platting for culture until the third passage. Thereafter, cells were harvested and further enriched by magnetic-activated cell sorting using rabbit-anti-CD90 antibody. After confirmation of metabolic viability (MTT-assay) and cluster-forming ability (crystal violet staining) of CD90+ cmGCs, the cells were labeled with a lipophilic red-fluorescent dye (PKH26) before transplanted into the recipient male goats by injection directly into the mediastinum testes under USG guidance. The colonization and repopulation of transplanted CD90+ cmGCs into the recipient ST was observed up to 8 weeks post-transplantation. The PKH26-labeled donor cell-derived colonies were identified in enzymatically digested ST and cryosections of recipient testes. Moreover, histochemical analyses revealed the restoration of the normal architecture of ST of recipient testis after GC transplantation. Therefore, the results suggest that the reproductive competence of infertile animals can be restored through mGC therapy and thus the methodology presented herein could be useful to obtain donor mGCs-derived functional male gametes in the recipient animal testis.

Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4-6 months) and adult (~ 2 years) bucks during non-breeding (July-August; n = 4 each) and breeding (October-November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and ß1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00515-x.

Diabetes Mellitus-A Risk Factor for the Development of Lumbar Disc Degeneration: A Retrospective Study of an Indian Population.

STUDY DESIGN: A retrospective study. OBJECTIVES: To determine the association between type-2 diabetes mellitus (T2DM) and the severity of lumbar disc degeneration disease (LDDD). METHODS: We included 199 patients with low back pain (LBP) who visited our hospital from 2016 to 2018. All patients were divided into 3 groups as per inclusion criteria. Group A, patients without DM (n = 75); group B, patients with controlled DM (n = 72); and group C, patients with uncontrolled DM (n = 52). The patients were further subdivided into group B1, DM duration ≤10 years (n = 38); group B2, DM duration >10 years (n = 34); group C1 DM duration ≤10 years (n = 28); and group C2, DM duration >10 years (n = 24). Sex, age, body mass index, occupation, smoking history, alcohol use, and duration of T2DM were recorded. The severity of LDDD was evaluated using the 5-level Pfirrmann grading system. Operated patients' disc materials were sent for histological examination. RESULTS: Demographic data showed no difference among groups (P > 0.5), except age. Patients with DM showed more severe disc degeneration compared with patients without DM. The average Pfirrmann scores between groups A and B1 had no difference; groups B2, C1, and C2 showed higher average Pfirrmann scores than group A (P < 0.05). Groups B2 and C2 showed higher average Pfirrmann scores than groups B1 and C1 (P < 0.05). Groups C1 and C2 showed higher average Pfirrmann scores than groups B1 and B2 (P < 0.05). The severity of LDDD was significantly related to DM duration in both groups B and C (P < 0.05). DM groups showed increased disc apoptosis and matrix aggrecan fragmentation, disc glycosaminoglycan content and histological analysis were significantly different; the results are similar to Pfirrmann score results. CONCLUSIONS: DM duration >10 years and uncontrolled DM were risk factors for LDDD.

Diabetes Mellitus and the Development of Lumbar Canal Stenosis: Is There Any Relevance?

STUDY DESIGN: Retrospective study. PURPOSE: To assess the relationship between the severity of lumbar canal stenosis (LCS) and type-II diabetes mellitus (DM). OVERVIEW OF LITERATURE: DM is a multiorgan disorder that has an effect on all types of connective tissues. LCS is a narrowing of the spinal canal with nerve root impingement that causes neurological claudication and radiculopathy. Identification of the risk factors of LCS is key in the prevention of its onset or progression. METHODS: LCS patients were divided into three groups as per DM status: group A without DM (n=150); group B patients with well-controlled DM; and group C patients with uncontrolled DM. Groups B and C were subdivided into group B1: patients with DM with a duration of ≤10 years (n=76), group B2: DM with duration of >10 years (n=68), group-C1 DM duration ≤10 years (n=56), and group C2 DM duration >10 years (n=48). The severity of LCS was evaluated using the Swiss Spinal Stenosis Scale (SSSS) and Modified Oswestry Disability score (MODS). Operated patients ligamentum flavum sent for histological staining and quantitative immunofluorescence analysis. RESULTS: The demographic data of groups did not show any difference except in age. There was no difference between the mean SSSS and MODS of groups A and B1. Groups B2, C1, and C2 had higher average SSSS and MODS than group A (p<0.05). Groups B2 and C2 had higher SSSS and MODS than groups B1 and C1. Groups C1 and C2 had higher scores than groups B1 and B2 (p<0.05). The severity of LCS was significantly related to the duration of DM in groups B and C (p<0.05). Uncontrolled and longer duration of DM had significant elastin fibers loss and also higher rate of disk apoptosis, high matrix aggrecan fragmentation, and high disk glycosaminoglycan content. CONCLUSIONS: Longer duration and uncontrolled diabetes were risk factors for LCS and directly correlate with the severity of LCS.

Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-ß1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (ß1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (ß-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and ß-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Dissociation of the Glenosphere: A rare Post-operative Complication of Reverse shoulder Replacement.

INTRODUCTION: Reverse shoulder replacement (RSR) has been accepted as the treatment of choice for glenohumeral arthritis with irreparable rotator cuff tear. Dislocation has been a potential complication of RSR but glenosphere disengagement is a rare complication itself. There have been only few published reports of this complication in the literature. CASE REPORT: In this case report, we have presented a case of repeated disengagement of glenoid sphere post-RSR in a 72-year-old male retired army personnel operated with Zimmer Biomet comprehensive RSR design. CONCLUSION: In our case scenario, we postulate that soft-tissue interposition was the reason for disengagement during first episode and was successfully relocated after removal. However, the subsequent disengagement was due to improper seating of sphere due to large central screw. Revision to a smaller central screw size appears to be the definitive solution in such case.

Osteoporotic burst fracture-clinical, radiological and functional outcome of three-column reconstruction using single posterior approach (Instrumentation, Corpectomy, Arthroscope Assisted Transpedicular Decompression and Mesh Cage).

PURPOSE: To evaluate a novel effective procedure utilizing three-column reconstruction via a posterior approach with a technique that utilizes an arthroscope to visualize the anterior surface of the dura during decompression. METHODS: A Prospective Study. 80 Osteoporotic vertebral burst fracture patients with similar demographic data managed by three-column reconstruction through single posterior approach surgery: Pedicle screw fixation, Corpectomy, Arthroscope Assisted Transpedicular Decompression (AATD) and Fusion (Mesh Cage + Bone grafting). Preoperative and postoperative clinical parameters (Visual Analog Score VAS, swestry Disability Index ODI, neurlogy, radiological parameters and surgical variables were recorded analysed. RESULTS: No significant differences in demographic data. Significant improvement was noted in VAS (pre-operative, 7.90 ±0.60; final follow-up 2.90 ± 0.54) and ODI (preoperative, 77.10 ± 6.96; final follow-up 21.30 ± 6.70). Neurological improvement was noted in 74 patients (Frankel grade E) while six patients remained non-ambulatory (Frankel grade C). Significant improvement was noted in local kyphosis angle (preoperative, 22.14 ± 2.60; postoperative, 10.40 ± 1.40) with a 10% loss of correction (2.5 ± 0.90) at final follow-up. Implant failure in two patients and proximal junctional failure in two patients managed with revision surgery. No iatrogenic dural or nerve injury. CONCLUSIONS: Osteoporotic Burst fracture can be managed with single posterior surgery, three-column reconstruction with mesh cage. It provides a significant improvement in clinical, radiological and functional outcomes. The arthroscope can improve a surgeon's operative field and magnification thereby ensuring complete decompression without injuring the dura or spinal cord.

Hyperglycemia and its influence on development of lumbar degenerative disc disease.

STUDY DESIGN: A Retrospective observational study. OBJECTIVES: To determine the influence of hyperglycemia on severity of lumbar degenerative disc disease (LDDD). METHODS: We retrospectively included 199 patients with low back pain (LBP) who visited our tertiary care hospital from June 2016 to December 2018. All patients divided into three groups as per inclusion and exclusion criteria. Group-A had patients without DM (n = 75). Group B had well-controlled DM patients (n = 72) and Group-C had uncontrolled DM patients (n = 52). Group B and C subdivided according to dutation of DM. Group-B1 DM duration was ≤ 10 years (n = 38), Group-B2 DM duration was >10 years (n = 34), Group-C1 DM duration ≤10 years (n = 28), Group-C2 DM duration >10 years (n = 24). Sex, age, BMI, occupation, smoking history, alcohol use and duration of type-II DM were recorded. The severity of LDDD was evaluated using the five-level Pfirrmann grading system. Operated patient's disc material sent for histological examination. RESULTS: Patients with DM showed more severe disc degeneration compared to patients without DM. The average Pfirrmann scores between Groups A and B1 had no difference; Groups B2, C1, and C2 showed higher average Pfirrmann-scores than Group-A (p > 0.05). Group-B2 and Group-C2 showed higher average Pfirrmann-scores than Group-B1 and Group-C1 (p > 0.05). Group-C1 and Group-C2 showed higher average Pfirrmann-scores than Group-B1 and B2 (p > 0.05). The severity of LDDD was significantly related to DM duration both in groups B & C (p > 0.05). DM groups showed increased disc apoptosis and matrix aggrecan fragmentation, Disc glycosaminoglycan content and histological significantly different, the results are similar to Pfirrmann-score results. CONCLUSIONS: There is a positive relationship between diabetes and LDDD. A longer the duration and poor control of hyperglycemia could aggravate disc degeneration.

Effects of different concentration and combinations of cryoprotectants on sperm quality, functional integrity in three Indian horse breeds.

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4-6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.