Recombinant Human-like Collagen Research Articles

Temperature-Controlled Expression of a Recombinant Human-like Collagen I Peptide in Escherichia coli.

Collagen is the functional protein of the skin, tendons, ligaments, cartilage, bone, and connective tissue. Due to its extraordinary properties, collagen has a wide range of applications in biomedicine, tissue engineering, food, and cosmetics. In this study, we designed a functional fragment of human type I collagen (rhLCOL-I) and expressed it in Escherichia coli (E. coli) BL21(DE3) PlysS containing a thermal-induced plasmid, pBV-rhLCOL-I. The results indicated that the optimal expression level of the rhLCOL-I reached 36.3% of the total protein at 42 °C, and expressed in soluble form. In a 7 L fermentation, the yield of purified rhLCOL-I was 1.88 g/L. Interestingly, the plasmid, pBV220-rhLCOL-I, was excellently stable during the fermentation process, even in the absence of antibiotics. Functional analyses indicated that rhLCOL-I had the capacity to promote skin cell migration and adhesion in vitro and in vivo. Taken together, we developed a high-level and low-cost approach to produce collagen fragments suitable for medical applications in E. coli.

Expression, characterization, and application potentiality evaluation of recombinant human-like collagen in Pichia pastoris

Collagen is a biofunctional protein that has been widely used in many fields, including but not limited to biomedical, cosmetics and skin care, food, and novel materials. Recombinant collagen has great potential as an alternative to collagen extracted from animals because it avoids the immune response, and the yield and properties are stable. However, challenges remain in the industrial application of recombinant collagen, including improving the expression yield, reducing the cost of purification for industry and expanding applications. In this study, a cloning and recombination method was used to heterologously express the recombinant human-like collagen (RHLC) in Pichia pastoris GS115 using the pPIC9k expression vector. The RHLC expression titre was 2.33 g/L via a 5-L fermenter, and the purification was completed within 48 h and was 98% pure. The characteristics of RHLC were investigated. Furthermore, potential applications for RHLC were explored, such as basal collagen sponge preparation, forming films with chitosan and production of collagen hydrolysed peptides. RHLC has various potential applications due to its triple helical structure, thermostability, good biocompatibility and film-forming ability.Graphical

Treatment of Acute Wounds With Recombinant Human-Like Collagen and Recombinant Human-Like Fibronectin in C57BL/6 Mice Individually or in Combination.

Wound repair is accomplished by the interaction between the cells involved in the repair and the extracellular matrix (ECM). Collagen is the main component of ECM, which is involved in transduction of signal, transportation of growth factors and cytokines. Fibronectin (FN) is also an important ECM, which participates in the initiation of fibroblast cell (FC) and promotes adhesion, migration, proliferation and differentiation of target cells. Compared with natural protein, the recombinant protein prepared by artificial method has the advantages of poor immunogenicity, wide range of sources, low cost and high activity. In this study, we used recombinant human-like collagen (RHC) and recombinant human-like fibronectin (rhFN) to treat acute wounds in C57BL/6 mice individually or in combination, and explored their effects on wound healing. Our study confirmed that these two recombinant proteins could effectively promote the proliferation, migration and adhesion of FCs. Meanwhile, it could positively regulate the healing speed and quality of acute wounds, re-epithelialization, collagen deposition, inflammation and angiogenesis. Moreover, we proved that the combination of the two was better than the treatment alone. Consequently, it has a good prospect as a new tissue material in the field of skin repair.

Transforming Growth Factor-β3/Recombinant Human-like Collagen/Chitosan Freeze-Dried Sponge Primed With Human Periodontal Ligament Stem Cells Promotes Bone Regeneration in Calvarial Defect Rats.

Patients with a skull defect are at risk of developing cerebrospinal fluid leakage and ascending bacterial meningitis at >10% per year. However, treatment with stem cells has brought great hope to large-area cranial defects. Having found that transforming growth factor (TGF)-β3 can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), we designed a hybrid TGF-β3/recombinant human-like collagen recombinant human collagen/chitosan (CS) freeze-dried sponge (TRFS) loading hPDLSCs (TRFS-h) to repair skull defects in rats. CFS with 2% CS was selected based on the swelling degree, water absorption, and moisture retention. The CS freeze-dried sponge (CFS) formed a porous three-dimensional structure, as observed by scanning electron microscopy. In addition, cytotoxicity experiments and calcein-AM/PI staining showed that TRFS had a good cellular compatibility and could be degraded completely at 90 days in the implantation site. Furthermore, bone healing was evaluated using micro-computed tomography in rat skull defect models. The bone volume and bone volume fraction were higher in TRFS loaded with hPDLSCs (TRFS-h) group than in the controls (p < 0.01, vs. CFS or TRFS alone). The immunohistochemical results indicated that the expression of Runx2, BMP-2, and collagen-1 (COL Ⅰ) in cells surrounding bone defects in the experimental group was higher than those in the other groups (p < 0.01, vs. CFS or TRFS alone). Taken together, hPDLSCs could proliferate and undergo osteogenic differentiation in TRFS (p < 0.05), and TRFS-h accelerated bone repair in calvarial defect rats. Our research revealed that hPDLSCs could function as seeded cells for skull injury, and their osteogenic differentiation could be accelerated by TGF-β3. This represents an effective therapeutic strategy for restoring traumatic defects of the skull.

Fabricating a novel HLC-hBMP2 fusion protein for the treatment of bone defects

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

Hybrid Freeze-Dried Dressings Composed of Epidermal Growth Factor and Recombinant Human-Like Collagen Enhance Cutaneous Wound Healing in Rats.

Epidermal growth factor (EGF) is important for promoting skin repair and remodeling. Native collagen is also widely used as a scaffold for skin tissue engineering. The limitations of EGF include easy decomposition or inactivation, whereas native collagen is immunogenic and has poor solubility. Therefore, we constructed a freeze-dried dressing based on the recombinant human-like collagen (RHC) to act as a carrier for EGF (RHC/EGF freeze-dried dressing) and promote skin wound closure. Here, the freeze-dried dressing that combined EGF and RHC significantly enhanced the proliferation, adhesion, and spreading of NIH/3T3 fibroblasts and migration of HaCaT keratinocytes at the wound site. The physicochemical characteristics of the RHC/EGF freeze-dried dressing investigated using scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry revealed that it was a loose and porous cake that redissolved quickly. The molecular mechanisms involved in cell proliferation and angiogenesis were also assessed. The expression levels of the markers Ki-67, proliferating cell nuclear antigen, vascular endothelial growth factor, and cluster of differentiation 31 were significantly increased after treatment with the RHC/EGF freeze-dried dressing (P < 0.01, vs. RHC or EGF alone). This increase indicated that the RHC/EGF freeze-dried dressing significantly accelerated wound closure, re-epithelialization, and the orderly arrangement and deposition of collagen in the Sprague–Dawley rats with full-thickness skin defects. This work describes a significant step toward the development of wound environments conducive to healing, and the RHC/EGF freeze-dried dressing is a potential therapeutic strategy in wound management.

Preparation recombination human-like collagen/fibroin scaffold and promoting the cell compatibility with osteoblasts.

On this basis, a novel recombinant human-like collagen (RHLC)/silk fibroin scaffold material with high porosity and controllable aperture was prepared. The compatibility of osteoblasts (OB) with the blends was tested in vitro. The morphology, adhesion and growth of scaffold cells were observed by scanning electron microscope and laser confocal microscope. Extensive measurements, including 3-[4, 5-dimethylthiazole-2-acyl]-2, 5-diphenyl tetrabrominate assays, intracellular total protein content, and alkaline phosphatase activity assays were performed after 7 days of culture. Survival and protein content increased in RHLC/fibroin stents. LSCM and SEM results confirmed that the cells grew better in the mixed scaffolds than in the pure silk scaffolds, and showed that the cells were easy to adhere and diffuse in the RHLC/silk scaffolds. RHLC/silk fibroin scaffolds are promising biomaterials for bone tissue engineering.

Newly Designed Human-Like Collagen to Maximize Sensitive Release of BMP-2 for Remarkable Repairing of Bone Defects.

Designing the “ideal” hydrogel/matrix which can load bone morphogenetic protein-2 (BMP-2) in a low dose and with a sustained release is the key for its successful therapeutic application to enhance osteogenesis. The current use of natural collagen sponges as hydrogel/matrix is limited due to the collagen matrix showing weak mechanical strength and unmanageable biodegradability. Furthermore, the efficiency and safe dose usage of the BMP-2 has never been seriously considered other than purely chasing the lowest dose usage and extended-release time. In this paper, we customized a novel enzymatically cross-linked recombinant human-like collagen (HLC) sponge with low immunogenicity, little risk from hidden viruses, and easy production. We obtained a unique vertical pore structure and the porosity of the HLC, which are beneficial for Mesenchymal stem cells (MSCs) migration into the HLC sponge and angiopoiesis. This HLC sponge loading with low dose BMP-2 (1 µg) possessed high mechanical strength along with a burst and a sustained release profile. These merits overcome previous limitations of HLC in bone repair and are safer and more sensitive than commercial collagens. For the first time, we identified that a 5 µg dose of BMP-2 can bring about the side effect of bone overgrowth through this sensitive delivery system. Osteoinduction of the HLC-BMP sponges was proved by an in vivo mouse ectopic bone model and a rat cranial defect repair model. The method and the HLC-BMP sponge have the potential to release other growth factors and aid other tissue regeneration. Additionally, the ability to mass-produce HLC in our study overcomes the current supply shortage, which limits bone repair in the clinic.

Physicochemical properties and biological behavior of injectable crosslinked hydrogels composed of pullulan and recombinant human-like collagen

Injectable hydrogels have become a hot topic of research in tissue engineering field, with broad application prospects. In this paper, recombinant human-like collagen (HLC) and three different kinds of high molecular weight pullulan (P), crosslinked with 1,2,7,8-diepoxyoctane (DEO), were synthesized in a water bath. Pullulan/1,2,7,8-diepoxyoctane (PD) and pullulan human-like collagen/1,2,7,8-diepoxyoctane (PHD) hydrogels, with high crosslinking density, injectability, thermal stability, and biocompatibility were synthesized. According to hypothesis, the biocompatibility and biodegradability of PHD hydrogels was better than PD hydrogels and PHD hydrogels served as ideal filler biomaterials in tissue engineering. Physicochemical properties such as swelling ratio, elastic modulus, thermal stability, porosity, and rheological features of these hydrogels were studied. Results showed that an increase in the molecular weight of pullulan and the amount of added HLC enhanced its mechanical properties. However, it showed little influence on its thermal stability, since pullulan and HLC themselves were thermally stable. Acute toxicity test, hemolysis test, and L929 cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay (MTT) were employed for testing the toxicity and cytocompatibility. The cell attachment in vitro and biocompatibility in vivo were also investigated. The MTT and live/dead cell viability staining results demonstrated that PDH was less cytotoxic than PD due to the promotion of the L929 proliferation. In vitro degradation results indicated that the hydrogels had prolonged degradation times and excellent anti-enzymatic abilities. Subcutaneous injection of PDH hydrogel in rabbit showed a lower inflammatory response after 1, 2, and 8 weeks, and the hydrogels showed almost no obvious degradation in two months. Thus, the hydrogel had a great potential to be used as ideal filler biomaterial in tissue engineering.

Improving osteogenesis of three-dimensional porous scaffold based on mineralized recombinant human-like collagen via mussel-inspired polydopamine and effective immobilization of BMP-2-derived peptide

An ideal bone substitute should be biocompatible, biodegradable, osteoinductive and osteoconductive. In our previous work, we fabricated a three-dimensional porous scaffold based on mineralized recombinant human-like collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA). Like other HA/collagen scaffolds, the nHA/RHLC/PLA scaffold lacked osteoinductive bioactivity. The purpose of the present study was to develop a polydopamine (pDA)-assisted BMP-2-derived peptide (designated as P24) surface modification strategy for improving the osteogenesis of the nHA/RHLC/PLA scaffold. The immobilization efficiency and release kinetics of P24, and in vitro osteoinductive activity of the nHA/RHLC/PLA-pDA-P24 scaffold were examined. The in vivo osteoinductive activity of the scaffold was evaluated usinga rat criticalsize calvarial defect model. Our results showed that pDA-assisted surface modification could more efficiently mediate the immobilization of P24 peptide onto the scaffold surfaces than physical adsorption. The in vitro release study showed that the P24 peptide was released slowly and steadily from the nHA/RHLC/PLA-pDA-P24 scaffold in a sustained manner, with a short initial burst release only during the first day, while the physisorbed nHA/RHLC/PLA-P24 group showed a sharp burst P24 release followed by a plateau phase. In vitro osteogenesis assay, the ALP activitiy and mRNA expression of osteo-specific markers of rat-derived mesenchymal stem cells (rMSCs) in the nHA/RHLC/PLA-pDA-P24 group were significantly higher than those of the nHA/RHLC/PLA-P24 and non-P24-loaded nHA/RHLC/PLA groups. In vivo, three-dimensional CT evaluation and histological examination demonstrated the nHA/RHLC/PLA-pDA-P24 scaffolds significantly enhanced bone regeneration of rat cranial defects to a much greater extent than physisorbed nHA/RHLC/PLA-P24 and non-P24-loaded nHA/RHLC/PLA scaffolds. Our findings indicated that the pDA-assisted surface modification method could significantly improve the osteogenesis activity of the nHA/RHLC/PLA scaffold and the new nHA/RHLC/PLA-pDA-P24 scaffold was a promising scaffold biomaterial for bone tissue regeneration.

A Two-Step Protocol to Remove Endotoxins from Human-Like Collagen

A two-step protocol to remove endotoxins from recombinant human-like collagen was investigated. To meet the clinical need, Triton X-114 two-phase extraction system was applied as the first step to remove large amounts of endotoxins, while affinity chromatography was used as the second step to achieve further purification. Under the best experimental condition optimized through response surface methodology techniques, endotoxin level was 0.025˜0.25 EU/mg and protein recovery was 81.9%, respectively, which indicated that 99% of endotoxins were eliminated with high protein recovery. Therefore, the two-step protocol can be exploited as an efficient method for endotoxin elimination in biotechnology processes.

Endotoxin Removal from Recombinant Human-like Collagen Preparations by Triton X-114 Two-phase Extraction

Endotoxin Removal from Recombinant Human-like Collagen Preparations by Triton X-114 Two-phase Extraction

Improved productivity of recombinant human-like collagen ii by supplying amino acids encoded by rare codons

Amino acids, the key growth factors, affect cell growth and metabolism directly or indirectly. In order to enhance the production of human-like collagen (HLC) II, amino acids were added into initial fermentation broth and response surface methodology (RSM) was used to screen the suitable varieties of amino acids and optimize their doses. Single-factor experiment showed that arginine, proline, threonine, cysteine and methionine could increase the production of human-like collagen II by 23.32 to 66.95%. With the optimization method of RSM, the final optimal concentration of arginine, cysteine and methionine were confirmed to be 5.95, 8.73 and 0.35 g·L-1 respectively. Under the optimal cultivation condition the maximal production of HLC reached 0.586 g·L-1 which was 150.43% higher than that of the control group at 0.234 g·L-1, while the acetate accumulation was 12.51% less than that of the control. Key words: Human-like collagen, methionine, rare condons, recombinant Escherichia coli, response surface methodology.

Improved productivity of recombinant human-like collagen ii by supplying amino acids encoded by rare codons

The spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has become a challenge to the global tuberculosis control and prevention program.Nevertheless, no adequate information has been made available on the drug susceptibility status of M. tuberculosis in Ethiopia. Hence, this study aimed atevaluating the drug resistance patterns of M. tuberculosis in the three sites of Ethiopia and its association with socio-demographic factors. A total of 102 M. tuberculosis isolates obtained from tuberculosis (TB) patients from three different sites were tested for drug susceptibility against the first and second-line anti-TB drugs using the proportion method on enriched Middle Brook 7H10 agar. The result showed that 36.3% of the isolates were resistant to at least one of the first line anti-TB drugs used in this study. The proportions of mono-resistance were 29.4, 13.7, 13.7 and 23.5% to isoniazid, rifampicin, streptomycin and ethambutol, respectively. The proportion of MDR M. tuberculosis was 11.8%. Only 55% of the isolates were susceptible to all the second line anti-TB drugs used in this study. One isolate (1%) was extensively drug-resistant (XDR). The occurrence of MDR M. tuberculosisisolates was not associated with sex, age, anti-TB treatment history, HIV status andlocation. The findings of this study showed moderate number of MDR M. tuberculosis isolates in the study area, warranting the necessity of the surveillance and monitoring. Key words: Mycobacterium tuberculosis, drug resistance, Ethiopia

Coordination study of recombinant human-like collagen and zinc (II)

In the present investigation, the complex of recombinant human-like collagen (r-HLC) with zinc (II) has been synthesized in aqueous solution and was analyzed by UV–vis spectroscopy, FTIR spectroscopy, circular dichroism (CD) spectroscopy, thermo gravimetric (TG) and differential scanning calorimetry (DSC) analysis. It can be concluded from UV–vis spectra that there exists interaction between r-HLC and zinc, and the complex is a new chemical compound different from pure r-HLC. In the complex of Zn, recombinant human-like collagen acts as ligand, linking the zinc ion via both groups of C O and N–H. Besides, the results of TG and DSC confirm that the complex was significantly different from ligand, and the former is more thermally stable in comparison with the latter. The results obtained from the current investigation are of crucial importance to understand the r-HLC–Zn complex and provide theoretical evidence for the further study.

A genetic algorithm for the optimization of the thermoinduction protocol for high‐level production of recombinant human‐like collagen from Escherichia coli

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.

The enhancement of osteogenesis by scaffold based on mineralized recombinant human-like collagen loading with rhBMP-2

A biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared with recombinant human bone morphogenic protein-2 (rhBMP-2) for improving the osteoinductive property of the scaffold. The nHA/RHLC/PLA scaffolds loaded with 10 μg rhBMP-2 and the unloaded scaffolds were implanted subcutaneously in the rat model. The osteogenetic capacity of these composites was evaluated by CT scan, ALP activity test and histological observation at 4 and 8 weeks after implantation. The experimental results indicated that the osteogenic capability of the scaffolds loaded with rhBMP-2 was superior to the unloaded scaffold. It was concluded that rhBMP-2 can enhance the osteoinductive property of the nHA/RHLC/PLA scaffold and the nHA/RHLC/PLA scaffold loaded with rhBMP-2 have the good potential of being used in bone tissue engineering.

Production of biomass and recombinant human-like collagen in Escherichia coli processes with different CO2 pulses

Production of biomass and recombinant human-like collagen in Escherichia coli processes with different CO2 pulses

Preparation and ectopic osteogenesis in vivo of scaffold based on mineralized recombinant human-like collagen loaded with synthetic BMP-2-derived peptide

The ideal bone graft material must be biocompatible, biodegradable, osteoconductive and osteoinductive. In this study, a new biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared and the synthetic P24 peptide derived from BMP-2 was introduced into the porous nHA/RHLC/PLA scaffold to improve its osteoinductive property. The nHA/RHLC/PLA implants loaded with 3 mg, 2 mg, 1 mg and 0 mg P24 peptide were implanted subcutaneously into rats. At the 4th, 8th and 12th weeks after implantation, the rats were sacrificed in batch and the samples were harvested. Their osteogenic capability was detected by CT scan and histological observation. The results indicated that the osteogenic capability of 3 mg, 2 mg and 1 mg of the P24 peptide was superior to the implants without the P24 peptide. There was no significant difference between implants with 3 mg and 2 mg P24 peptide, but the osteogenic capability of the two dosage groups was significantly better than that of the 1 mg group. It was concluded that BMP-2-derived peptide can increase the osteoinduction of nHA/RHLC/PLA scaffold and the P24 peptide induced new bone formation in a dose-dependent manner. The nHA/RHLC/PLA scaffold loaded with the synthetic BMP-2-derived peptide is a kind of ideal scaffold material for bone tissue engineering.

Production of biomass and recombinant human-like collagen in Escherichia coli processes with different CO2 pulses

The evolution of CO(2) in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O(2)-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO(2) concentration of 12.7% in the effluent gas was detected. The CO(2) pulse injection experiments showed that: (1) a 20% CO(2) pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO(2) inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO(2) pulse. The higher the CO(2) concentration and/or the longer the duration of the CO(2) pulse, the stronger the stimulatory or inhibitory effects.