Amnion and chorion matrix maintain hMSC osteogenic response and enhance immunomodulatory and angiogenic potential in a mineralized collagen scaffold

Abstract

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

Evaluating osteogenic effects associated with the incorporation of ascorbic acid in mineralized collagen scaffolds

“…Porosity was calculated as the fraction of the scaffold weight change over the theoretical maximum amount of IPA, a product of the density of IPA (0.786 mg mm À3 ) and the calculated volume of the dry scaffold. 30 Porosity ¼ Wet weight ½ ÀDry weight ½…”

“…Lifted hMSCs were seeded onto hydrated scaffolds at a total density of 100,000 cells per scaffold (150,000 cells per scaffold for RNA isolation) using a previously established protocol. 30,31,49,50 First, 50,000 cells (5 μL of a 10,000 cells/μL growth media cell solution) were seeded on one side of the scaffold and incubated for 30 min.…”

“…Two different protocols were used for RNA isolation (n = 5), based on previously established methods. 27,29,30 For cultured cells in suspension (Day 0 RNA), an RNAqueous™-Micro Total RNA Isolation Kit (ThermoFisher Scientific) was used, with no deviations from the provided protocol. For seeded scaffolds (Day 3, 7, 14, 28), a Qiagen™ RNeasy Kit with a modified lysis protocol was employed.…”

“…The degree of mineral deposition within the scaffold was quantified using a previously described method. 28,30,31 Briefly, cell-seeded scaffolds (n = 6) were cultured with phenol-red-free growth media to Day 14 or Day 28, and an unseeded group of scaffolds was used as a Day 0 control. After the designated culture period, scaffolds were rinsed with PBS and fixed with Epredia™ Formal-Fixx™ (ThermoFisher Scientific) overnight.…”

“…For example, incorporation of zinc, different glycosaminoglycans, amnion or chorion matrix, and even manuka honey have all been shown to alter scaffold mechanical properties or cell behavior. [28][29][30][31] Noncytokine-associated strategies to modify scaffold composition and boost biological activity are particularly exciting in regard to clinical translation, and we now aim to broaden our exploration further with the consideration of ascorbic acid.…”