Recombinant Human Basic Fibroblast Growth Research Articles

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy.

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications. Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model. Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model. Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

Clinical efficacy of recombinant human basic fibroblast growth factor combined with ranitidine in the treatment of recurrent oral ulcer and its effect on serum TNF, IL-2 and T-lymphocyte subsets.

To evaluate the clinical efficacy of recombinant human basic fibroblast growth factor (rh-bFGF) combined with ranitidine in the treatment of recurrent oral ulcer and its effects on serum TNF, IL-2 and T-lymphocyte subsets. This was a retrospective study. Eighty patients with oral ulcers admitted to First Medical Center, Chinese PLA General Hospital from July 2021 to June 2022 were randomly divided into the control group and the experimental group (n=40). Patients in the control group were given topical treatment with rh-bFGF gel, while those in the experimental group were given oral treatment combined with ranitidine based on the control group, and both groups were treated continuously for 14 days. The therapeutic effect, pain relief time, ulcer healing time, as well as the differences in the levels of inflammatory factors and T-lymphocyte subsets were compared and analyzed between the two groups. The overall response rate of the experimental group was 92.5%, while that of the control group was 75%, with a statistically significant difference(P=0.03). After treatment, inflammatory factors indexes in the experimental group were significantly lower than those in the control group, with statistically significant differences (P=0.00). The indexes of T-lymphocyte subsets in the experimental group were significantly higher than those in the control group after treatment, with statistically significant differences (P=0.00). Recombinant human basic fibroblast growth factor combined with ranitidine is effective in the treatment of recurrent oral ulcers, boasting various benefits such as effectively promoting ulcer healing, reducing pain and inflammatory response, and enhancing immune function.

Application value of bone cement containing rhbFGF and RHBMP-2 in PKP treatment of osteoporotic lumbar compression fracture

To investigate the effect of bone cement containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human bone morphogenetic protein-2 (rhBMP-2) in percutaneous kyphoplasty(PKP)treatment of osteoporotic vertebral compression fracture(OVCF). A total of 103 OVCF patients who underwent PKP from January 2018 to January 2021 were retrospectively analyzed, including 40 males and 63 females, aged from 61 to 78 years old with an average of (65.72±3.29) years old. The injury mechanism included slipping 33 patients, falling 42 patients, and lifting injury 28 patients. The patients were divided into three groups according to the filling of bone cement. Calcium phosphate consisted of 34 patients, aged(65.1±3.3) years old, 14 males and 20 females, who were filled with calcium phosphate bone cement. rhBMP-2 consisted of 34 patients, aged (64.8±3.2) years old, 12 males and 22 females, who were filled with bone cement containing rhBMP-2. And rhbFGF+rhBMP-2 consisted of 35 patients, aged (65.1±3.6) years old, 14 males and 21 females, who were filled with bone cement containing rhbFGF and rhBMP-2. Oswestry disability index (ODI), bone mineral density, anterior edge loss height, anterior edge compression rate of injured vertebra, visual analog scale (VAS) of pain, and the incidence of refracture were compared between groups. All patients were followed for 12 months. Postoperative ODI and VAS score of the three groups decreased (P<0.001), while bone mineral density increased (P<0.001), anterior edge loss height, anterior edge compression rate of injured vertebra decreased first and then slowly increased (P<0.001). ODI and VAS of group calcium phosphate after 1 months, 6 months, 12 months were lower than that of rhBMP-2 and group rhbFGF+rhBMP-2(P<0.05), bone mineral density after 6 months, 12 months was higher than that of rhBMP-2 and group calcium phosphate(P<0.05), and anterior edge loss height, anterior edge compression rate of injured vertebra of group rhbFGF+rhBMP-2 after 6 months and 12 months were lower than that of group rhBMP-2 and group calcium phosphate(P<0.05). There was no statistical difference in the incidence of re-fracture among the three groups (P>0.05). Bone cement containing rhbFGF and rhBMP-2 could more effectively increase bone mineral density in patients with OVCF, obtain satisfactory clinical and radiological effects after operation, and significantly improve clinical symptoms.

Transplantation of Induced Pluripotent Stem Cells (iPSCs) into Cuprizone Model of Demyelination in Rat

Introduction: Multiple Sclerosis (MS) is a neurodegenerative inflammatory disease with a wide range of axonal demyelination and sensory-motor disorders. Today, stem cell therapy is an appropriate choice for MS management. Induced Pluripotent Stem Cells (iPSC) are pluripotent stem cells that have neuroprotective effects with neuroglial differentiation potential. In this study, we used iPSCs to improve remyelination and behavioral function in model of cuprizone multiple sclerosis disease in rats. Method: Human iPSCs were cultured and extended on a mouse embryonic fibroblast feeder cell layer inactivated with mitomycin-C in DMEM/F12 supplemented with 20% knockout serum replacement, 0.1 mmol/L nonessential amino acids, and 10 ng/mL of recombinant human basic fibroblast growth factor in a 5% CO2 and 95% humidity. The demyelination model was induced using a 0.2 oral Cuprizone regime. Thereafter, iPSCs were transplanted after six weeks. Remyelination was investigated via histological assessments and immunocytochemistry following six-week post-transplantation. The functionality was evaluated using behavioral tests, BBB, and Footprint following six-week post-transplantation. Results: The results of in vivo studies showed that after specific myelin tissue staining, the cuprizone diet led to the induction of demyelinated areas in the brain tissue six weeks after the cuprizone diet. Differentiation of transplanted cells was confirmed via the immunocytochemistry technique of PLP. The results of the Footprint test showed that the motor function of the paws of the animals improved compared to the control group. Moreover, BBB behavioral tests showed improved symptoms in the experimental groups that received the cells. Conclusion: In total, iPSCs can improve remyelination and functional recovery following transplantation into the Cuprizone demyelination model in rats. Therefore, iPSC therapy could improve behavioral function in multiple sclerosis disease

Long Term Culture of Mesenchymal Stem Cells: No Evidence of Chromosomal Instability

Introduction: Mesenchymal stem cells (MSCs) technology has opened promising roads toward different aspects of medicine and biology. However, possible tumorgenic potential of MSCs has raised many concerns in using them in regenerative medicine. In the present study, we aimed to investigate the safety of MSCs by in-vitro culture and karyotype analysis. Materials and Methods: MSCs were successfully cultured in DMEM medium including 3.5 ng/ml recombinant human basic fibroblast growth factor (bFGF) and 20 % fetal bovine serum (FBS) within 25 passages. Results: No significant abnormality was observed in basic karyotype and morphology of MSCs in different passages of culture. Conclusions: Using of EGF free medium containing low bFGF concentration are possible explanations for observed integrity in karyotype and morphology of MSCs. Our findings would be further convincing evidence on the safety of MSCs under suggested conditions to guarantee using of them in regenerative medicine and disease treatment.

Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers

A suitable microenvironment or niche is essential for self-renewal and pluripotency of stem cells cultured in vitro, including bovine embryonic stem cells (bESCs). Feeder cells participate in the construction of stem cell niche by secreting growth factors and extracellular matrix proteins. In this study, metabolomics and transcriptomics analyses were used to investigate the effects of low-density feeder cells on bESCs. The results showed that bESCs co-cultured with low-density feeder cells experienced a decrease in pluripotent gene expression, cell differentiation, and a reduction of central carbon metabolic activity. When cell-permeable pyruvate (Pyr) and recombinant human basic fibroblast growth factor (rhbFGF) were added to the culture system, the pluripotency of bESCs on low-density feeder layers was rescued, and acetyl-coenzyme A (AcCoA) synthesis and fatty acid de novo synthesis increased. In addition, rhbFGF enhances the effects of Pyr and activates the overall metabolic level of bESCs grown on low-density feeder layers. This study explored the rescue effects of exogenous Pyr and rhbFGF on bESCs cultured on low-density feeder layers, which will provide a reference for improvement of the PSC culture system through the supplementation of energy metabolites and growth factors.

Application of recombinant human basic fibroblast growth factor in repair of the aged dog bite wound

BackgroundDog bite wounds usually have severe local soft tissue damage and the wounds are likely to be contaminated by a large number of bacteria or even a series of rare pathogenic bacteria, leading to repeated infection and delayed wound healing. Recombinant human basic fibroblast growth factor (rh-bFGF) has excellent ability to promote wound repair and widely used in various complicated wounds. ObjectiveObserve the clinical efficacy of Recombinant human basic Fibroblast growth factor (rh-bFGF) applicated in the treatment of dog bite wounds. MethodsA total of 31 patients (16 adult and 15 aged) with rabies exposure grade Ⅲ dog bite wounds treated in the department of emergency surgery in our hospital from February 2020 to February 2022 were reviewed. Rh-bFGF was applied in the wounds after debridement and the patients were treated with preventive anti-inflammatory therapy. Observe the wound healing rules, infection rate, related complications and application time of antibiotics. ResultsEventually, all the wound were well healed and the healing time needed for most patients were about 2–3 weeks. The wound infection rate of aged patients was much higher, therefore application time of intravenous antibiotics injection in the aged patients was significantly higher than it in the adults (P < 0.05), but the difference of the time reach the intermittent wound dressing change was not statistically significant (P > 0.05). ConclusionRh-bFGF is safe and reliable for severe dog bite wounds in both the elderly and adults, it may help improve the early stage healing speed of aged patients, but the specific treatment needs to be individualized and accurate depends on different personal physique and wound conditions.

Effect of the combination of composite skin grafting and recombinant human basic fibroblast growth factor on plastic surgery for extensive sc ars after burns

Purpose: To investigate the effect of combined use of composite skin grafting and recombinant human basic fibroblast growth factor (rh-bFGF) on plastic surgery for extensive burn scars, and its influence on wound healing and inflammatory responses in patients.&#x0D; Methods: Hospital records of 120 patients who underwent extensive scar plastic surgery after burns in Hanchuan People’s Hospital from February 2019 to February 2020 were retrospectively analyzed. The patients were equally assigned to study group (STG) and control group (COG), based on the order of admission. All patients received composite skin grafting surgery and post-surgery topical silver sulphadiazine cream regularly, while those in study group (STG) received additional treatment with rh-bFGF spray on the wound surfaces. Scar scores, degree of wound healing, populations of fibrocytes and capillaries, levels of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1), and activities of lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were evaluated.&#x0D; Results: Patients in STG had significantly better scar scores, shorter mean wound healing time, higher mean degree of wound healing, higher scores on inflammatory reactions near the wounds, and higher populations of fibrocytes and capillaries, when compared with patients in COG group (p &lt; 0.001). After surgery, there were lower levels of VEGF and TGF-β1, and lower levels of activities of LDH and SDH in STG than in COG group (p &lt; 0.001).&#x0D; Conclusion: Rh-bFGF accelerates the proliferation of wound tissue, improves local microcirculation, regulates inflammatory response, and enhances wound healing in burn scar patients when used in combination with composite skin grafting. Therefore, Rh-bFGF has a potential for enhanced management of burn scars.

Implanted synthetic cells trigger tissue angiogenesis through de novo production of recombinant growth factors.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106 protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane-like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Therapeutic potential of recombinant human basic fibroblast growth factor on postoperative patients with chronic rhinosinusitis with nasal polyps.

To explore the effect of intranasal administration of recombinant human basic fibroblast growth factor (rh-bFGF) on postoperative chronic rhinosinusitis with nasal polyps (CRSwNP) patients. A prospective, randomised, controlled, single-blinded trial. Seventy-five hospitalised patients who met the criteria of primary bilateral CRSwNP were enrolled from March 2020 to January 2021. Visual analogue scale, 22-item Sino-Nasal Outcome Test, Lund-Kennedy (L-K) system and scanning electron microscopy and quantitative real-time polymerase chain reaction. Seventy-five patients with CRSwNP were randomly assigned to three groups, and 72 patients completed the 1-month medication regimen and 1-year follow-up. Rh-bFGF nasal-spray and drop application reduced general nasal VAS scores within 2 weeks after endoscopic sinus surgery (ESS) compared to the control group. In contrast, only rh-bFGF nasal-drops reduced SNOT-22 scores at 2 weeks and 1year compared with the control group. A significant reduction in the endoscopic L-K score was observed in the rh-bFGF nasal-spray and drop group compared with the control group. This is primarily because rh-bFGF promotes cilia growth in the nasal mucosal epithelium after the operation, as illustrated by scanning electron microscopy and expression of CP110, Tap73 and Foxj1 mRNA. For eosinophilic CRSwNP, the general VAS score of rh-bFGF nasal-drops was more obviously reduced compared to the control group after ESS. A similar trend was observed for L-K score. Rh-bFGF nasal-drops and sprays can quickly and effectively relieve postoperative symptoms and improve long-term prognosis of patients with CRSwNP. Moreover, rh-bFGF nasal-drops is also an effective method for postoperative patients with eosinophilic CRSwNP.

Effects of methacrylic anhydride gelatin hydrogel loaded with silver and recombinant human basic fibroblast growth factor on deep partial-thickness burn wounds in rabbits

Effects of methacrylic anhydride gelatin hydrogel loaded with silver and recombinant human basic fibroblast growth factor on deep partial-thickness burn wounds in rabbits

Fibroblast Growth Factor-Impregnated Collagen-Gelatin Sponge Improves Keratinocyte Sheet Survival.

Commercially available cultured epithelial keratinocyte sheets (KSs) have played an essential role in wound healing over the past four decades. Despite the initial uptake by the dermal elements, the survival rate of KS on the dermis-like tissue generated by conventional artificial dermis (AD) is low, making this method unsuitable for standard treatments. Therefore, an innovative AD such as collagen-gelatin sponge (CGS) that maintains the release of human recombinant basic fibroblast growth factor (bFGF) may promote wound healing. In this study, we examined whether combination therapy with KSs and CGS with bFGF (bFGF-CGS) could enhance KS survival by heterologous grafting by transplantation of human-derived KSs in an athymic nude rat wound model of staged skin reconstruction. The CGSs were implanted into skin defect wounds on athymic nude rats, which were then divided into two experimental groups: the bFGF group (CGSs containing bFGF, n = 8) and the control group (CGSs with saline, n = 8). Two weeks after implantation, human epithelial cell-derived KSs were grafted onto the dermis-like tissue, followed by assessment of the survival and morphology at 1 week later using digital imaging, histology (hematoxylin and eosin and Masson's trichrome staining), immunohistology (von Willebrand factor), immunohistochemistry (cytokeratin 1-5-6, Ki-67), and immunofluorescence (collagen IV, pan-cytokeratins) analyses. The bFGF group showed a significantly higher KS survival area (86 ± 58 mm2 vs. 32 ± 22 mm2; p < 0.05) and increased epidermal thickness (158 ± 66 μm vs. 86 ± 40 μm; p < 0.05) compared with the control group, along with higher dermis-like tissue regeneration, neovascularization, epidermal maturation, and basement membrane development. These results indicate that the survival rate of KSs in the dermis-like tissue formed by bFGF-CGS was significantly increased. Therefore, combination treatment of bFGF-CGS and KSs shows potential for full-thickness skin defect reconstruction in clinical situations. Impact statement This study highlights how using a combination of cultures, keratinocyte sheets, and collagen-gelatin sponge containing basic fibroblast growth factors can significantly improve cell survival in athymic nude rats with staged skin reconstruction. Our study makes a significant contribution to the literature because it highlights a novel and improved strategy for treating a very common condition such as skin wounds arising from many conditions. Clinical translation of this study may be useful for treating skin wounds.

The therapeutic effects of basic fibroblast growth factor in nasal vestibulitis

ObjectiveTo observe the effect of topical application of recombinant human basic fibroblast growth factor (bFGF) in patients with nasal vestibulitis. MethodsOne hundred patients with nasal vestibulitis were randomly divided into two groups. Local application of bFGF + conventional medication was administered in the treatment group, while conventional medication was conducted in the control group. The healing of the nasal vestibular mucosa was observed. ResultsThe mucosal healing time was 18.3 ± 4.8 days in the treatment group and 36.2 ± 6.2 days in the control group. The data comparison revealed that the difference between groups was statistically significant (P < 0.01). The total effective rate was 98.0% in the treatment group and 90.0% in the control group, and the difference was not statistically significant between the two groups (P > 0.05). ConclusionTopical application of bFGF in patients with nasal vestibulitis could promote the growth of nasal mucosa, shorten the healing time of mucosal erosion, enhance the clinical treatment effect, and save a lot of treatment time for patients.

Stabilisation of Recombinant Human Basic Fibroblast Growth Factor (FGF-2) against Stressors Encountered in Medicinal Product Processing and Evaluation

Basic fibroblast growth factor (FGF-2) is a highly labile protein with strong potential for tissue engineering. The aim of this study was to develop FGF-2 formulations that are stable against physical stressors encountered in pharmaceutical processing and evaluation. Pharmaceutical excipients, alone or in combination, were added to aqueous FGF-2 (770 ng/mL) solution and the stability of the resulting solutions on storage at 4–37 °C was evaluated. Stability of the solutions to repeated freeze-thaw cycles and lyophilisation was also evaluated, as well as the stability of the lyophilised stabilised protein to storage at −4, 4 and 18 °C for up to 12 months. In all of these experiments FGF-2 was quantified by ELISA assay. The as-received FGF-2, when dissolved in water, was highly unstable, retaining only 50% of baseline protein content after 30 min at 37 °C or 1 h at 25 °C. By contrast, FGF-2 solutions prepared with 0.5% w/v methylcellulose (MC) and 20 mM alanine (formulation F5) or with 0.5% w/v MC and 1 mg/mL human serum albumin (HSA) (formulation F6) were highly stable, having residual FGF-2 content comparable to baseline levels even after 2 h at 37 °C and 5 h at 25 °C. F5 and F6 were also highly stable to repeated freeze-thaw cycles, with >99% of FGF-2 load remaining after the third cycle. In addition, F5 and F6 were stable to lyophilisation, and the lyophilised products could be stored at −4, 4 or 18 °C for at least 12 months, with less than 1% loss in mean FGF-2 content. Thus, FGF-2 solution is effectively stabilised against both thermal and processing stressors in the presence of MC and alanine (F5), or MC and HSA (F6). The resultant FGF-2 solutions may be applied as medicinal products or further processed into more advanced medicinal products, e.g., scaffolds, for wound healing and tissue regeneration.

The effects of recombinant human basic fibroblast growth factor on nerve regeneration in a partial defect inferior alveolar nerve model in rabbits

ObjectiveThe purpose of this study was to investigate the effect of recombinant human basic fibroblast growth factor (rhbFGF) on nerve regeneration immediately after nerve injury. MethodPartial defects were created in the inferior alveolar nerve of rabbits and were treated with (Experimental group) or without (Control group) rhbFGF for 1–3 weeks. Injured tissues were then characterized immunohistochemically using primary antibodies to S-100; a marker for Schwann cells, and to neurofilament (NF); a marker for axons. ResultsThe number of S-100-immunopositive cells in the experimental group was significantly increased compared with the control group at weeks 2 and 3 (P < 0.05). The number of NF-immunopositive cells in the experimental group was also significantly increased compared with the control group at week 3 (P < 0.05). ConclusionThese results suggest that application of rhbFGF immediately after partial damage of an inferior alveolar nerve might be effective to promote nerve regeneration.

Application of bioactive hydrogels combined with dental pulp stem cells for the repair of large gap peripheral nerve injuries

Due to the limitations in autogenous nerve grafting or Schwann cell transplantation, large gap peripheral nerve injuries require a bridging strategy supported by nerve conduit. Cell based therapies provide a novel treatment for peripheral nerve injuries. In this study, we first experimented an optimal scaffold material synthesis protocol, from where we selected the 10% GFD formula (10% GelMA hydrogel, recombinant human basic fibroblast growth factor and dental pulp stem cells (DPSCs)) to fill a cellulose/soy protein isolate composite membrane (CSM) tube to construct a third generation of nerve regeneration conduit, CSM-GFD. Then this CSM-GFD conduit was applied to repair a 15-mm long defect of sciatic nerve in a rat model. After 12 week post implant surgery, at histologic level, we found CSM-GFD conduit could regenerate nerve tissue like neuron and Schwann like nerve cells and myelinated nerve fibers. At physical level, CSM-GFD achieved functional recovery assessed by a sciatic functional index study. In both levels, CSM-GFD performed like what gold standard, the nerve autograft, could do. Further, we unveiled that almost all newly formed nerve tissue at defect site was originated from the direct differentiation of exogeneous DPSCs in CSM-GFD. In conclusion, we claimed that this third-generation nerve regeneration conduit, CSM-GFD, could be a promising tissue engineering approach to replace the conventional nerve autograft to treat the large gap defect in peripheral nerve injuries.

Phenotypic instability of mesenchymal stromal cells isolated and expanded in different growth media

Background & Aim Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are an attractive candidate for use in regenerative therapies and are being widely investigated for the treatment of a broad spectrum of diseases. Despite this, the efficacy of treatment is unproven and interpretation of trial data is difficult, in part due to the heterogeneity of MSC preparations as a result of variable manufacturing processes. MSCs are currently isolated in basal media containing fetal bovine serum (FBS), platelet lysate or other supplements but little is understood about the impact of changes in media composition on the biology, therapeutic action or potency of MSCs. The aim of this study was to analyse the influence of widely used culture media on the growth parameters, differentiation potential and surface marker expression of human BM-MSCs. Methods, Results & Conclusion Whole bone marrow obtained from the iliac crest of a 24-year-old healthy donor was plated in a standard basal medium with 5 different supplements: (1) a proprietary serum-free supplement, (2) 5% human platelet lysate, (3) 10% FBS, screened and proven to support MSC isolation and expansion (4) 10% screened FBS and 1 ng/ml recombinant human basic fibroblast growth factor (FGF2) and (5) 10% unscreened FBS and 1 ng/ml FGF2. The impact of media composition on growth kinetics, morphology, differentiation propensity and surface immunophenotype was assessed. The latter involved the BD Lyoplate flow cytometry screening panel of 242 monoclonal antibodies. MSC growth kinetics were highly influenced by expansion medium, with the greatest proliferation rate observed in MSCs cultured in the serum-free supplemented medium. Cell morphology varied greatly between cell preparations. Adipogenesis and osteogenesis were maintained in all culture conditions, but the degree of differentiation varied between conditions. Chondrogenesis was most starkly effected by changes in expansion medium and was only observed in serum-containing media. Analysis of surface marker expression revealed significant variation in response to culture conditions, with 70 of the 242 markers differentially expressed between cell preparations. These data suggest that changes in the composition of the culture media – for example switching batches of FBS – have profound effects on key biological attributes of BM-MSCs and on their surface marker expression profiles. This demonstrates the critical need for harmonisation of media formulation to aid interpretation of clinical results.

Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85 g/L in the shake flask and 7.7 g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.

Epidermal Necrosis of Forearm and Wrist due to Extravasation of Sodium Bicarbonate and Application of Epidermal Growth Factor for Therapy in Extremely Low Birth Weight Infant

Extravasation occurs when an intravenous drug is inadvertently administered into the perivascular space or subcutaneous tissue. This can have various consequences, from local irritation to severe tissue loss. Extravasation occur more frequently in neonatal populations, especially in younger gestational ages and with lower birth weights. There is no established method of treatment for extravasation. Conservative treatments such as saline wash, wet dressing, hyaluronidase, and hydrogel dressing should be performed. Recently, two full-term newborns have been reported to successfully treat skin necrosis with recombinant human basic fibroblast growth factor. We report for the first time that extravasation in extremely very low birth weight infants can be successfully treated with recombinant human epidermal growth factor.

Observation of curative effect of recombinant human basic fibroblast growth factor combined with compound polymyxin B ointment and local application of insulin on wound healing of deep second-degree burn in diabetes mellitus: a randomized study.

We aimed at investigating the clinical efficacy of recombinant human basic fibroblast growth factor combined with compound polymyxin B ointment and the local application of insulin on wound healing of deep second-degree burn in diabetes mellitus. 80 diabetes mellitus patients complicated with deep second-degree burn treated from 2016 to December 2017 were selected and divided into observation group (n=40) and control group (n=40) using the random number method. Patients in control group were treated with compound polymyxin B ointment, while those in observation group were treated with basic fibroblast growth factor based on the treatment in control group. The time of wound pain relief, wound scarring, and wound healing was compared. The number of positive bacteria in the wound was recorded and the duration of infection was also recorded. Moreover, the changes in the expressions of Advanced Glycation End products (AGEs) and Vascular Endothelial Growth Factor (VEGF) were analyzed. In observation group, the time of wound pain relief, wound scarring, and wound healing was significantly shorter than that in control group (p<0.05). At 3, 14, and 28 days after the treatment, the levels of AGEs in observation group were lower than those in control group, while the levels of VEGF in the observation group were higher than those in control group (p<0.05). The proportions of wound infection, skin necrosis, systemic blood infection, hypoglycemia, and hyperglycemia and the ineffective rate in observation group were significantly lower than that in control group (p<0.05). The application of compound polymyxin B ointment and the local application of insulin combined with recombinant human basic fibroblast growth factor in diabetes mellitus patients complicated with a deep second-degree burn can effectively alleviate the pain, reduce the wound infection rate, promote the wound healing, and improve the overall therapeutic effect.