The Collagen-targeting Parathyroid Hormone-related Peptide Promotes Collagen Binding And In Vitro Chondrogenesis From Bone Marrow-derived MSCs

Background:Recent years, there are more and more degenerative joint diseases such as lumbar discherniation (LDH) and degenerative osteoarthritis caused by articular cartilage degenerativeand defect. These degenerative joint diseases of articular cartilage affect more than a thirdof the world population. Surgical techniques for the treatment of cartilage defects have beenextensive research and development. However, until now, there is no treatment canfundamentally cure the loss of biological function caused by disc degeneration. Thereforeoptimized treatment strategies for articular cartilage lesions are of a high socio-economicimportance. With the research and development of tissue engineering of orthopedics, itoffers a new ideal approach for the treatment of degenerative joint disease.Intervertebral disc tissue engineering is still in its infancy, and the exploration of disctissue engineering mainly focuses on three parts: seeding cells, scaffolds and growth factors.Bone marrow derived mesenchymal stem cells (Bone marrow mesenchymal stem cells,BM-MSCs) has been widely used as a common seed cells in tissue engineering. Duringtissue engineering, the decay of seed cells, degenerating of seed cells with passaged, theloss of biology function of degeneration seed cells are the major difficulties. In order toovercome these difficulties, a large number of exogenous growth factors have been added tothe scaffolds to maintain the seed cell biology function. However in practice, factor simplydelivered in solution is difficult to be retained in the scoffolds due to its rapid diffusion inextracellular fluids and cannot promote the seed cells persistence. Collagen, especially typeI collagen is the most commonly used tissue engineering scaffolds, so it is an importanttarget of tissue engineering materials targeting.Parathyroid hormone-related protein (Parathyroid hormone related protein, PTHrP) is identified and purified as a factor involved in human hypocalcaemia of malignancy in1987.It is composed by141amino acid chloride. There is a high homology between itsN-terminal PTHrP (1-34) and the N-terminal parathyroid hormone (N-PTH) amino acidsequence. Apart from similar physiological activity of N-PTH, PTHrP also has otherbiological functions, such as proliferating chondrocytes and inhibits chondrocytedifferentiation toward hypertrophy in the growth plate through the PTHrP-Indian hedgehog(IHH) axis. Meanwhile, the collagen binding domain (collagen-binding domain, CBD),which consists of a sequence of seven amino acids (TKKTLRT),and takes a role of collagenbinding, has been widely used for binding the associated active molecules to collagen.Objectives:In this study, we build a new biologically active recombinant protein CBD-PTHrP byrecombinant DNA technology, it has a dual biological function: on the one hand, PTHrPcould induce bone marrow mesenchymal stem cells into cartilage differentiation and inhibitchondrocyte differentiation toward hypertrophy, on the other hand CBD sequence couldbind to the collagen scaffold material, so that the PTHrP play a lasting effect on the seedcells. Therefore, new bioactive factor CBD-PTHrP, building by recombinant DNAtechnology, is used to solve aging of seed cells in the experiment degeneration, and lay thefoundation for building a new type of intervertebral disc.Methods:1. The expression and purification of CBD-PTHrP recombinant protein, and theidentification of collagen target capacity.CBD coding sequence was constructed to the both ends of the primers, and cDNA wasused as a template. The coding gene sequence of CBD-PTHrP and NAT-PTHrP protein wasacquired by PCR, and then inserted into the prokaryotic expression vector pET-32a. Therecombinant plasmid was transformed into TOP10competent cells and the positive cloneswere screened and identified, and then the positive plasmid was transformed into theRosetta (DE3) competent cells. IPTG was used to induce CBD-PTHrP and NAT-PTHrPprotein expression, and the protein was purified by Ni-column.0,0.5,1,2,4,8,12,16μmfusion protein were added to the96well plate coated with collagen, collagen binding capacity of the protein was detected by ELISAs.2. Human bone marrow mesenchymal stem cells were isolated, cultured andindentified.Human bone marrow mesenchymal stem cell was isolated from the human bonemarrow by gradient centrifugation. The cells were cultured with DMEM/F12+10%FBSmedium, and the flow cytometry was used to detect the molecular markers of theBM-MSCs: CD34, CD44, CD45, CD73, CD90, CD105, when the cells transmitted to thethird generation.3. CBD-PTHrP promotes chondrocyte differentiation and inhibits chondrocytedifferentiation toward hypertrophy from BM-MSCs.The pellet was made from BM-MSCs, and cultured with cartilage inducing medium.The medium was changed every2-3days. Two weeks later,100ng/ml NAT-PTHrP andCBD-PTHrP protein was added to the medium separately, and a blank control group isnecessary. Two weeks after that, total RNA and protein was extracted and Real-time PCRand Western blot was used to detect the expression of COL1A1, COL2A1, COL10A1andSox-9at mRNA and protein level. The pellet was also embedded by paraffin. Safranin-Ostaining was used to detect the cartilage formation, and the COL1A1, COL2A1, COL10A1and Sox-9expression in the pellet was measured by immunohistochemical staining.Results:1. pET-32a-NAT-PTHrP and pET-32a-CBD-PTHrP recombinant plasmid areconstructed, and the protein of CBD-PTHrP and NAT-PTHrP are induced and purified.Compared with NAT-PTHrP protein, CBD-PTHrP protein has better collagen bindingcapacity.2. Human BM-MSCs is isolated and indentified, the results show that the immunemarkers for CD34and CD45are negative, excluding the divided cells are hematopoieticcells or leukocytes; while CD44, CD73, CD90and CD105surface antigen are positive, toindentify the cells is the BM-MSCs.3. Real-time PCR and Western blot results indicate that, compared with the controlmedium (CM) group, CBD-PTHrP and NAT-PTHrP protein can significantly induced COL2A1and Sox-9gene expression, and inhibit COL1A1and COL10A1gene expressionat the mRNA and protein levels. Safranin-O staining demonstrate that compared with theCM group, CBD-PTHrP and NAT-PTHrP induce cartilage cell formation in the BM-MSCscell pellet. The immunohistochemical detection results also indicate that the CBD-PTHrPand NAT-PTHrP can induce the COL2A1and Sox-9protein expression and inhibitCOL1A1and COL10A1protein expression, which is consistent with the experimentalresults of the Real-time PCR and Western blot.Conclusion:Compared with the NAT-PTHrP protein, CBD-PTHrP protein has bettercollagen-binding capacity by the use of CBD peptide. It indicates that CBD-PTHrP couldbind collagen I scoffolds more tightly in vitro and in vivo and continue promoting seed celldifferentiation. At the same time, CBD-PTHrP gets the same biological activity ofNAT-PTHrP. It could induce chondrocytes from BM-MSCs and inhibit chondrocytedifferentiation toward hypertrophy. These results indicate that adding CBD peptide at theN-terminus of PTHrP has no effect on the biological activity. Moreover, CBD peptidepromotes PTHrP binding collagen I scoffolds for a long time. Therefore it could continueinducing chondrocytes from BM-MSCs and inhibiting chondrocyte differentiation towardhypertrophy. In conclusion, our study suggests CBD-PTHrP could be an efficient system fortargeting cartilage tissue engineering in clinical application.