A Novel Technique for Tissue Engineering Periosteum Using Three-Dimensional Bioprinting

Background

Periosteum plays a vital role in bone repair by housing a population of cells with remarkable regenerative capacity, including osteoblasts and mesenchymal stem cells. Periosteal flaps are often used by reconstructive surgeons to promote healing of bone defects caused by trauma, congenital deformities, or tumor resection. However, the utility of periosteal flaps is limited by donor-site availability and morbidity. This study presents a unique method for periosteum tissue engineering in which a three-dimensional (3D) bioprinter was used to print a precise pattern of periosteal derived cells (PDCs) onto collagen scaffolds.

 

Methods

PDCs were isolated from the periosteum of bovine femurs and expanded in culture. PDCs were then mixed with alginate gel to create a bio-ink, which was printed in three different experimental groups: bio-ink alone; bio-ink printed on a type I collagen (COL1) scaffold; and bio-ink printed on a type II collagen (COL2) scaffold. To maximize the viability of the PDCs, the bio-ink was printed in a precise lattice pattern¹ that was created using computer-aided design (CAD) software. PDCs were also cultured in monolayer (no alginate, no collagen) to serve as a control group. All groups were incubated in culture media and evaluated at one and two week time points. Live/Dead staining was used to assess cell viability. Polymerase chain reaction (PCR) was used to quantify gene expression and assess osteogenic differentiation.

 

Results

On gross examination, the COL1 and COL2 scaffold groups maintained greater structural integrity than the bio-ink only group. Live/Dead imaging showed high viability of cells at one and two weeks in all experimental groups. PCR results demonstrated an increase in gene expression of the osteogenic differentiation markers osteocalcin (OCN) and alkaline phosphate (ALP) in all treatment groups relative to the monolayer control group. OCN expression was most significant in the COL1 group. PCR also showed an increase in COL2 gene expression in all treatment groups, but most significantly in the COL2 scaffold group.

 

Conclusions

The results presented here support a novel method for using 3D bioprinting to engineer periosteum constructs. The COL1 and COL2 scaffolds promoted cell viability and structural stability. Increases in OCN and ALP gene expression suggest the PDCs were undergoing osteogenic differentiation, with the COL1 scaffold being most supportive of this phenotype. The increase in COL2 gene expression, a chondrogenic marker, suggest some of the PDCs may be undergoing early chondrogenic differentiation before endochondral ossification into bone. In vivo studies are currently underway to assess the capacity of this tissue engineered periosteum to induce bone repair in an animal model.

 

References

1. Jia J, Richards DJ, Pollard S, et al. Engineering alginate as bioink for bioprinting. Acta biomaterialia. 2014;10(10):4323-4331. doi:10.1016/j.actbio.2014.06.034.