Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Uso de los factores de crecimiento presentes en el plasma rico en plaquetas como un tratamiento alternativo de lesiones músculo esqueléticas en animales / Use of growth factors present in platelet-rich plasma as an alternative treatment for musculoskeletal lesions in animals / Uso de fatores de crescimento presentes no plasma rico em plaquetas como tratamento alternativo para lesões musculo esqueléticas em animais

Resumen Se realizó una revisión sistemática del uso de plasma rico en plaquetas (PRP) en animales, seleccionando 105 artículos de referencia, publicados en los últimos 8 años (2012 a 2020) de las revistas Veterinary Medicine International, Scientific Reports, BMC Veterinary Research, Platelets, Biomedicine and Pharmacotherapy entre otras y validadas en las siguientes bases de datos: Pubmed, Web of Science, Sciencedirect, Scielo, Medline, Embase y Scopus. Limitando solo las revistas con altos rankings ayudados con las clasificaciones de revistas por scimago journal. Solo 35 artículos cumplieron los criterios de selección; articulándolo con necesidades actuales en la medicina veterinaria, buscando una nueva biotecnología y proyectándose hacia la medicina regenerativa, basándose en reconstrucción de tejidos vivos para contrarrestar daño o remplazo de función de órganos lesionados. Una de estas herramientas es el plasma rico en plaquetas (PRP) con sus factores de crecimiento (FC) Transformante ß (TGF ß), fibroblástico básico (FGFb), derivado de plaquetas (PDGF), del endotelio vascular (VEGF), tejido conectivo (CTGF) y epidérmico (EGF). Actualmente el (PRP) es un bioestimulante tomado como base por estudios en medicina humana y medicina veterinaria, observando su efectividad en el proceso de consolidación ósea en pacientes clínicos. Se recogieron evidencias científicas presentadas en literatura médica con respecto al PRP como una alternativa de tratamiento en el manejo de fracturas y lesiones músculo esqueléticas, con ello se logra determinar la importancia de la estandarización del procedimiento (PRP) en uso, preparación, almacenamiento y administración.

Abstract A systematic review of platelet-rich plasma (PRP) use in animals was carried out by taking 105 reference articles published during the last 8 years (2012 to 2020) in Veterinary Medicine International, Scientific Reports, BMC Veterinary Research, Platelets, Biomedicine and Pharmacotherapy; they were validated in the following databases: PubMed, Web of Science, ScienceDirect, SciELO, Medline, Embase and Scopus. The search was limited to just journals having a high impact factor; the SCImago journal rank (SJR) indicator (i.e. a sophisticated citation analysis algorithm) was also used. Only 35 of these articles met the selection criteria: i.e. coordinating content with current needs in veterinary medicine, seeking new biotechnological approaches and promoting regenerative medicine based on reconstructing living tissue to counteract damage or replace injured organs' function. One such tool is platelet-rich plasma (PRP) with its derivative growth factors (GF): transforming growth factor-ß (TGF-ß), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and epidermal growth factor (EGF). PRP is currently used as a biostimulant, acting as the basis for human and veterinary medicine studies; its effectiveness regarding bone/fracture healing/regeneration in clinical patients has been observed. Scientific evidence in the pertinent medical literature regarding PRP as an alternative treatment for managing fractures and musculoskeletal injuries was collected, thereby determining the importance of standardising the PRP procedure regarding its use, preparation, storage and administration.

Resumo Foi realizada uma revisão sistemática da utilização de plasma rico em plaquetas (PRP) em animais, com 105 artigos de referência, publicados nos últimos 8 anos (2012 a 2020) das revistas Veterinary Medicine International, Scientific Reports, BMC Veterinary Research, Platelets, Biomedicina e Farmacoterapia entre outros e validados nas seguintes bases de dados: Pubmed, Web of Science, Sciencedirect, Scielo, Medline, Embase e Scopus. Limitar apenas os periódicos com classificações altas ajudou com as classificações de periódicos pelo jornal scimago. Dos quais apenas 35 artigos atenderam aos critérios de seleção; articulando-se com as necessidades atuais da medicina veterinária, buscando uma nova biotecnologia e projetando-se na medicina regenerativa, baseada na reconstrução de tecidos vivos para neutralizar danos ou substituição de função de órgãos lesados. Uma dessas ferramentas é o plasma rico em plaquetas (PRP) com seus fatores de crescimento (FC) ß transformador (TGF ß), fibroblasto básico (FGFb), derivado de plaquetas (PDGF), endotelial vascular (VEGF), tecido conjuntivo (CTGF) e epidérmico (EGF). Atualmente (PRP) é um bioestimulante tomado como base para estudos em medicina humana e veterinária, observando sua eficácia no processo de consolidação óssea em pacientes clínicos. Foram coletadas evidências científicas apresentadas na literatura médica a respeito do PRP como alternativa de tratamento no manejo de fraturas e lesões musculoesqueléticas, determinando a importância da padronização do procedimento (PRP) no uso, preparo, armazenamento e administração.

Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Human bone morphogenetic protein-2 (hBMP-2) characterization by physical-chemical, immunological and biological assays.

Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor ß (TGF-ß) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.

Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays

Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor ß (TGF-ß) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.