Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients

Abstract

Tissue engineering‐based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite‐collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3‐year follow‐up. Among DEGs, osteogenesis‐related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in “in vitro” experiments with hASCs.

Enhancement of the Biological and Mechanical Performances of Sintered Hydroxyapatite by Multiple Ions Doping

“…Human adipose-derived mesenchymal stem cells (hASC) were purchased from Lonza Milan, Italy (Catalog n. PT-5006) as cryopreserved frozen cells at the first passage. These cells are positive for surface markers CD13, CD29, CD44, CD73, CD90, CD105, CD166, while are negative for other markers, such as CD14, CD31, CD45 (Mazzoni et al, 2020). Cells were expanded in Dulbecco's Modified Eagle Medium F-12 (DMEM/F12; Lonza, Milan, Italy), supplemented with 10% fetal bovine serum (FBS) and 10% antibiotics (Pen/Strep 10.000 U/ml) at a density of 5,000 cells/cm 2 , in a T75 flask (Falcon BD, Franklin Lakes, NJ, United States) at 37 • C with 5% CO 2 in a humidified atmosphere (Manfrini et al, 2013;Mazzoni et al, 2017Mazzoni et al, , 2020.…”

“…These cells are positive for surface markers CD13, CD29, CD44, CD73, CD90, CD105, CD166, while are negative for other markers, such as CD14, CD31, CD45 (Mazzoni et al, 2020). Cells were expanded in Dulbecco's Modified Eagle Medium F-12 (DMEM/F12; Lonza, Milan, Italy), supplemented with 10% fetal bovine serum (FBS) and 10% antibiotics (Pen/Strep 10.000 U/ml) at a density of 5,000 cells/cm 2 , in a T75 flask (Falcon BD, Franklin Lakes, NJ, United States) at 37 • C with 5% CO 2 in a humidified atmosphere (Manfrini et al, 2013;Mazzoni et al, 2017Mazzoni et al, , 2020. At the second passage, hASCs were randomly assigned to five treatment groups: hASCs grown in monolayer in 24-well tissue culture polystyrene plates (TCP) and hASCs grown on biomaterials, such as (i) S-HA, (ii) S-MgHA, (iii) S-MgSrHA, (iv) S-MgZnHA.…”

“…Plates were incubated at 37 • C. in humidified air (5% CO 2 ) until the time of the assay. RT 2 Profiler TM PCR Array was performed at day 14 (Mazzoni et al, 2020).…”

“…Human ASCs were grown on biomaterials and plastic plates employed as the control (TCPS) until day 14. After this period, total RNA was extracted through RNeasy Plus Micro Kit (Qiagen, Milan, Italy) according to the manufacturer's instructions (Mazzoni et al, 2020). RNA quality and quantity were assessed using a Nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, United States) and stored at -80 • C until the time of the analysis (Mazzoni et al, 2012).…”

“…RT 2 Profiler PCR TM Array PCR TM was performed using SYBR Green method on a CFX96 Touch PCR detection system (Bio-Rad, Milan, Italy) (Mazzoni et al, 2017). In terms of data analysis, fold-changes of each gene expression were calculated using the 2 − CT method, whereas the housekeeping genes employed as controls were used to normalize the results (Mazzoni et al, 2017(Mazzoni et al, , 2020. Two independent experiments were performed simultaneously.…”