New strategies for sterilization and preservation of fresh fish skin grafts.

The introduction of fish skin as a biological dressing for treating burns and wounds holds great promise, offering an alternative to existing management strategies. However, the risk of disease transmission is a significant concern. Therefore, this study aimed to examine how established sterilization and preservation procedures affected fish skin grafts' microbiological and histological properties for long-term usage. Lyophilization of the fish skin graft followed by rehydration in normal saline for 15 min did not change the collagen content. Furthermore, gamma irradiation of the lyophilized fish skin graft at different lengths 5, 10, and 25 KGy showed a significant reduction in microbial growth (aerobic bacteria, aerobic yeasts, and fungi) at 15- and 30 days after the irradiation. However, exposure to 10 KGy was found to be the most effective intensity among the different gamma irradiation lengths since it preserved the collagen fiber content and intensity in the lyophilized fish skin grafts at 15- and 30 days after the irradiation. These findings provide efficient preservation and sterilization methods for long-term usage of the fresh Tilapia skin grafts used for biological dressings.

In vitro expansion of human mesenchymal stem cells and hepatocytes using purified platelet derived growth factors, a potential therapeutic modality for liver fibrosis.

Mesenchymal stem cells (MSCs) and hepatocytes are considered valuable candidates for cell-based therapy. The use of free zoonotic media, as purified platelets derived growth factors (L-GF) and human platelet lysate (hPL), instead of using fetal bovine serum (FBS) to support the growth and proliferation of these cells could be used as a promising therapeutic tool in hepatic regeneration. This study aimed to evaluate the usage of purified platelet derived growth factors and platelet lysate in both MSCs and hepatocyte cultures and to compare them with the usage of FBS. MSCs and hepatocytes were cultured in growth media supplemented with L-GF or hPL and compared them to their culture in growth media supplemented with FBS. Cells were subjected to population doubling (PD) and generation time (GT) calculations. The best result for MSCs was that obtained by using 10% hPL or 10% FBS with the highest cell count, highest viability and shortest incubation time needed to reach confluency compared to supplementation with 10%, 20% or 30% L-GF. As for hepatocyte culture, the use of 10% FBS for supplementation of media used for hepatic cell proliferation showed better performance regarding cell count, viability, and incubation time to reach confluency compared to the use of either hPL or L-GF. In conclusion, our study showed that 10% hPL had the best results in MSCs culture which suggests that hPL could be a better alternative for the development of xenofree stem cell culture that can be used for many clinical applications. On the other hand, 10% FBS showed the best results in hepatocyte culture.

Lyophilized equine platelet-rich plasma (L-GFequina) antagonize the Reproductive toxicity and oxidative stress Induced by Cyclophosphamide in female rats.

BACKGROUND: The antineoplastic agent Cyclophosphamide (CP) induces reproductive toxicity. New strategies for protecting ovarian tissue damage in women with chemotherapy-induced reproductive toxicity are essential. This study was designed to evaluate the possible protective effect of combined treatment with L-GFequina on CP-induced reproductive toxicity in the mature female rat. METHODOLOGY: Forty mature female rats were assigned into four groups: First group, control: rats were intraperitoneally injected (IP) with 200 µl sterile saline solution on days 1 and 10; Group 2 (CP): were IP injected with 75 mg/kg on days 1 and 10 to induce POI); Group 3 (CP + L-GFequina): as in group 2 + IP injected with 200 µl rehydrated L-GFequina half-hour after CP injection on day 1 and 10); Group 4 (L-GFequina): rats were IP injected with 200 µl L-GFequina on day 1 and 10). Blood samples were collected for a complete blood picture and determinations of nitric oxide and malondialdehyde. Animals were sacrificed on Day-21, and genitalia was dissected, weighed, and fixed in 10% formalin for histopathological and morphometric evaluation. RESULTS: On day 21 of the experiment, body weight, ovarian parameters (Ovarian weight, uterine weight, the number of ovarian follicles, and corpora lutea (CL) were determined, and histopathological changes, blood profile, as well as antioxidant activity assessment, were performed. CP significantly suppresses ovarian and uterine functions and increased MAD, NO levels, RBCs, hemoglobin, WBCs, and platelet count compared to the control group ( P < 0.05). While, in CP + L-GFequina group, gross, histomorphometry parameters, blood, and biochemical markers were similar to that in the control. IP injection of L-GFequina alone significantly (P < 0.05) increased body weight, and ovarian and uterine morphometry compared with the control. CONCLUSION: co-administration of L-GFequina with CP might protect the reproductive organs in rats through its high antioxidant capacity.

Effectiveness of lyophilized growth factors injection for subacromial impingement syndrome: a prospective randomized double-blind placebo-controlled study.

BACKGROUND: Platelet-derived lyophilized growth factors (L-GFs) use a standardized number of allogenic pathogen-free platelets instead of autologous platelets used in PRP as a source of growth factors. This study aimed to evaluate the efficacy of L-GF injection versus placebo in subacromial impingement (SIS) treatment. METHODS: The current randomized double-blind placebo-controlled study included sixty patients (40 females and 20 males, aged between 24 and 75 years) diagnosed with SIS (both clinically and sonographically). Patients were randomly assigned to two equal groups. Under ultrasound guidance, group 1 received subacromial saline injection, and group 2 received L-GF injection. Clinical examination, pain visual analogue scale (VAS), shoulder pain and disability index (SPADI) and shoulder ultrasound were performed before and at the 8th week after injection. RESULTS: Follow-up assessment showed statistically significant improvement in the L-GF group regarding active flexion, active and passive internal rotation and extension, SPADI-disability scale, VAS and thickness of the supraspinatus tendon by US. Regression analysis showed that group 1 was approximately 30 times more likely than the L-GF group to experience painful arc at follow-up. Both groups showed statistically significant improvement in SPADI-pain scale and SPADI-total, flexion and abduction (still the mean value of abduction was significantly higher in the L-GF group). CONCLUSIONS: L-GF injection resulted in clinically significant reductions in pain and functional disability outcomes in patients with SIS. An objective significant reduction in the thickness of the supraspinatus tendon, measured by ultrasound, in the L-GF group hopefully encourages proper healing and functioning in SIS. TRIAL REGISTRATION: The identification number is NCT04330027, date of first registration (01/04/2020). Unique on 21/11/2019, Protocol ID: 0106178.

Intraovarian Injection of Reconstituted Lyophilized Growth-Promoting Factor Extracted from Horse Blood Platelets (L-GFequina) Increases Oocytes Recovery and In Vitro Embryo Production in Holstein Cows.

The purpose of this study was to determine the impact of intraovarian injections of a reconstituted lyophilized growth-promoting factor extracted from horse blood platelets (L-GFequina) on the number of ovarian follicles, the recovery of cumulus−oocyte complexes (COCs), and embryo development to the blastocyst stage in Holstein cows. Thus, 12 Holstein cows were assigned to three protocols. According to the number of punctured follicles in protocol 1, ovum pick-up (OPU) was conducted on days 6 and 14 of the cycle (day 0 = estrus). In protocol 2, every large follicle (more than 7 mm) was removed, and 1 mL of L-GFequina was intraovarian injected (day 0). Two days later, equine chorionic gonadotropin (eCG) was administered, and OPU sessions were conducted on days 6, 10, and 14. The same ovarian stimulation procedure as that in protocol 2 was performed in protocol 3, except that equine L-GFequina was not supplied. OPU was carried out on days 6 and 10 of the cycle. The results indicate that the intraovarian injection of L-GFequina significantly (p < 0.05) increased the number of OPU sessions per cycle, the recovery of cumulus−oocyte complexes (COCs), and the production of blastocysts. In conclusion, an intraovarian injection of L-GFequina can improves OPU-IVEP results in Holstein cows.

Repair of experimentally induced femoral chondral defect in a rabbit model using Lyophilized growth promoting factor extracted from horse blood platelets (L-GFequina).

Lyophilized equine platelet derived growth factors (LGF) is a novel advanced platelet rich protein growth factor. It has been successfully applied in various fields of regenerative medicine to treat a variety of inflammatory and degenerative musculoskeletal conditions. Our study aimed to evaluate the efficacy of intraarticularly injected LGF for the remedy of articular cartilage injury, commonly characterized by progressive pain and loss of joint function in osteoarthritic rabbits. Full-thickness cylindrical cartilage defects were generated in both femoral condylar articular surfaces in twenty rabbits. The left joint of all animals was injected with the adjuvant as a self-control negative, while the right joint was injected by LGF. Four- and eight-weeks post-surgery, the femoral condyles were harvested, and assessed grossly, microscopically and immunohistochemically. Cytokines (TNF-α, IL-1ß, PDGF and TGF-ß1) contents of the chondral defects were quantified by ELISA as well as the gene expression of Col I and Col II via RT-qPCR. The LGF treated defects showed significant higher ICRS (International cartilage repair society) healing scores of cartilaginous regeneration with a significant higher histological healing score on using O'Driscoll histological scoring system. Additionally, LGF significantly lowered the levels of the pro-inflammatory cytokines TNF-α and IL-1ß. It also significantly increased the anabolic and angiogenic growth factors (PDGF and TGF-ß1), and significantly elevated the expression of chondrogenic-related marker genes; Col I and Col II. The current study reveals that LGF improves chondral healing and thus it can be a superior nominee as an adjunctive therapy to positively influence regeneration of chondral defects in osteoarthritic patients.

Safety and immunogenicity evaluation of inactivated whole-virus-SARS-COV-2 as emerging vaccine development in Egypt.

BACKGROUND: Current worldwide pandemic coronavirus disease 2019 (COVID-19) with high numbers of mortality rates and huge economic problems require an urgent demand for safe and effective vaccine development. Inactivated SARS-CoV2 vaccine with alum. Hydroxide can play an important role in reducing the impacts of the COVID-19 pandemic. In this study, vaccine efficacy was evaluated through the detection of the neutralizing antibodies that protect mice from challenge with SARS-CoV 2 3 weeks after the second dose. We conclude that the vaccine described here has safety and desirable properties, and our data support further development and plans for clinical trials. METHODS: Characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069; and MW250352 at GenBank were isolated from Egyptian patients SARS-CoV-2-positive. Development of inactivated vaccine was carried out in a BSL-3 facilities and the immunogenicity was determined in mice at two doses (55 and 100 µg per dose). RESULTS: The distinct cytopathic effect induced by SARS-COV-2 propagation on Vero cell monolayers and the viral particles were identified as Coronaviridae by transmission electron microscopy and RT-PCR on infected cells cultures. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless of the dose concentration, with excellent safety profiles and no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by the wild virus challenge of the vaccinated mice and viral replication detection in lung tissues. CONCLUSIONS: Vaccinated mice recorded complete protection from challenge infection via inhibition of SARS-COV-2 replication in the lung tissues of mice following virus challenge, regardless of the level of serum neutralizing antibodies. This finding will support future trials for the evaluation of an applicable SARS-CoV-2 vaccine candidate.

Effect of Intrauterine Infusion of Equine Fresh Platelets-Rich Plasma (PRP) or Lyophilized PRP (L-GFequina) on Ovarian Activity and Pregnancy Rate in Repeat Breeder Purebred Arabian Mares.

This study was designed to examine the effect of the intrauterine infusion of platelet-rich plasma (PRP) or equine lyophilized growth factor (L-GFequina) on the follicular growth, endometrial thickness, estrus cycle length, and pregnancy rate in purebred Arabian mares. A total of 73 purebred Arabian mares who experienced repeat breeding for three successive cycles were randomly divided into the following three groups: control group, without treatment; second Group (PRP group), in which mares were intrauterine infused with 20 mL of fresh PRP on the second day after the end of physic estrus phase; and the third group (L-GFequina Group), consisting of mares that were intrauterine infused with 20 mL of reconstituted lyophilized horse platelets growth factors (L-GFequina) on the second day after the estrus phase. The results showed no significant difference between control and treated groups in the diameter of the preovulatory follicles during the post treatment cycle. The endometrium thickness increased significantly in the L-GFequina and PRP groups ahead of the non-treated group. Intrauterine L-GFequina or PRP administration shortened the estrus cycle length. A higher pregnancy rate was recorded in the L-GFequina and PRP treated mares. In conclusion, the intrauterine infusion of L-GFequina or PRP increased the endometrial thickness and pregnancy rate and could be used to improve fertility in Arabian purebred mares who experienced from repeat breeding.

Using intra-articular allogenic lyophilized growth factors in primary knee osteoarthritis: a randomized pilot study.

Objective: Investigating the safety in addition to clinical and structural efficacy of allogenic lyophilized growth factors (L-GFs) in patients with symptomatic primary knee osteoarthritis. Design: A prospective, open-label pilot study. A total of 31-patients randomized into non-intervention and intervention groups. Materials & methods: The intervention group received two intra-articular doses at baseline and after 2-months. Post-injection complications were documented, and the efficacy was assessed by Western Ontario and McMaster Universities Osteoarthritis Index scores and ultrasonography. Results: One dropout from the intervention group. The percentage of improvement of mean Western Ontario and McMaster Universities Osteoarthritis Index-scores and ultrasonography-detected effusion were statistically significant in the intervention group compared with the non-intervention. A brief, mild, post-injection pain was reported by all intervention group. Conclusion: This study provides the safety of intra-articular injection of allogenic L-GFs in knee osteoarthritis. The conclusion of efficacy was limited by small sample size and lack of control injection. Clinical trial registration: NCT04331327 (ClinicalTrials.gov, retrospectively registered).

Lay abstract Knee osteoarthritis affects many people after middle age. It causes progressive joint damage and may lead to disability. To date, there is no medical treatment that can stop disease progression and restore knee function. In this study, we tested a novel treatment for knee osteoarthritis. Patients who received knee injection with growth factors showed better results in joint pain and swelling. Growth factors were isolated from donated platelets and were assured that they were free of pathogens.

Lyophilized growth factor intralesional injection in female pattern hair loss: A clinical and trichoscopic study.

Various therapeutic modalities have been tried for female pattern hair loss (FPHL) treatment. To our knowledge, no previous studies had evaluated the therapeutic effect of lyophilized growth factor (L-GF) intralesional injection in FPHL. The current study aimed to evaluate the efficacy and safety of intralesional L-GF injection in FPHL by clinical and trichoscopic evaluation. This study included 20 patients with FPHL. All patients received three treatment sessions of intralesional injection of L-GF 4 weeks apart. Patients were followed-up for further 3 months. The outcome was evaluated by trichoscopy, photography score, patient's satisfaction score and side effects were reported. Trichoscopic evaluation showed significant posttreatment increase in all hair parameters associated with a significant decrease in vellus hair count. Ludwig's grade II showed posttreatment significant differences in all trichoscopic parameters from the baseline. No significant differences were detected regarding all trichoscopic parameters between the two Ludwig's grades posttreatment. 80% of patients showed photography score improvement that was significantly higher in Ludwig's grade II than in grade I. 100% of patients showed improvement in patient's satisfaction score with insignificant difference between Ludwig's grades. Intralesional injection of L-GF is safe and improved various trichoscopic hair parameters and clinical scores in FPHL.

Ablative fractional CO2 laser vs lyophilized growth factor intralesional injection vs combination of both modalities for striae distensae treatment.

BACKGROUND: Different studies had evaluated the efficacy of fractional CO2 laser and platelet-rich plasma (PRP) in treating striae distensae (SD). Lyophilized growth factors (LGF) represent another form of delivering growth factors similar to PRP with a more standardized method. To the best of our knowledge, no previous trials have been reported using LGF in SD. AIMS: We aimed to compare the efficacy of ablative fractional CO2 laser vs intralesional injection of LGF vs combination of both modalities for SD treatment. METHODS: This study included 20 female patients with SD. All patients received three modalities of treatment in separate three areas: area A: received fractional CO2 laser, area B: received combination of fractional CO2 laser and intradermal injection of LGF, and area C: received intradermal injection of LGF. Each area received three sessions with 6-week intervals. The outcome was evaluated clinically and histopathologically before treatment and six weeks after the last session. RESULTS: There was a statistically significant clinical and histopathological improvement of SD both in areas A and B after treatment. Area C exhibited nonsignificant clinical and histopathological improvement. Area B showed the best improvement results as compared to areas A and C. CONCLUSION: We reported that fractional CO2 laser combined with LGF injection was more effective than fractional CO2 laser alone in SD treatment based on clinical and histopathological assessment. We do not advocate LGF as a monotherapy for SD treatment; instead, LGF can be used as a combined therapy with fractional CO2 laser to improve its outcome.

The diagnostic value of anti-cyclic citrullinated peptide antibodies in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation of the joints. The aim of this study was to evaluate the diagnostic value of anti CCP antibodies in patients with RA. This study included three groups; group I: 60 early diagnosed RA patients selected according to the ACR, group II: 30 patients of arthritis other than RA, group III: 10 healthy age and sex matched volunteer. Sera from all participants were tested for rheumatoid factor (IgM RF) and anti-CCP IgG antibodies using ELISA. Of 60 patients in group I, 52 subjects (86.7%) were RF positive, whereas all participants in groups II and III were negative. Similarly 56 patients (93.3%) of group I were anti-CCP antibody positive, but all subjects in groups II and III were negative. Thus detection of anti-CCP antibodies can be used as a diagnostic indicator for patients with early RA (P < 0.0001).

Cyclosporine A sensitizes the kidney to tubulointerstitial fibrosis induced by renal warm ischemia.

BACKGROUND: Renal warm ischemic injury and immunosuppression with cyclosporin A (CsA) may contribute to chronic allograft nephropathy after cadaveric transplantation. This study establishes whether CsA can sensitize the kidney to injury and fibrosis induced by renal warm ischemia (RWI). METHODS: The left kidney of Sprague-Dawley rats was subjected to 30 min of warm ischemia and/or intraperitoneal CsA (15 mg/kg/d) for 30 days (n=6 per group). Renal injury and fibrosis were assessed histologically together with immunohistochemistry for collagen III, transforming growth factor (TGF)-beta1, ED1 (macrophage marker), and alpha-smooth muscle actin. Renal mRNAs for collagen III, TGF-beta 1, matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase-1 together with MMP enzyme activity were also determined. RESULTS: RWI or CsA alone produced only minor effects on renal injury and fibrosis. However, in CsA-treated rats, RWI produced a marked increase in tubulointerstitial fibrosis, as shown by the potentiation of collagen III and TGF-beta1 determined by immunochemistry and mRNA analysis. The up-regulation of tissue inhibitor of metalloproteinase-1 mRNA was associated with a decrease in MMP enzyme activity. In CsA-treated rats, RWI was also associated with an increase in inflammatory infiltrates, elevated immunostain for ED1 (indicating extensive macrophage influx), and elevated immunostain for alpha-smooth muscle actin (indicating myofibroblast activation). CONCLUSIONS: In the rat, CsA can sensitize the kidney to fibrosis induced by renal warm ischemia. In renal transplantation, when cadaveric donor kidneys have been subjected to a period of warm ischemia, CsA may be an inappropriate choice for immunosuppressive therapy.