Mechanical strain induces ex vivo expansion of periosteum

Abstract

Segmental bone defects present complex clinical challenges. Nonunion, malunion, and infection are common sequalae of autogenous bone grafts, allografts, and synthetic bone implants due to poor incorporation with the patient's bone. The current project explores the osteogenic properties of periosteum to facilitate graft incorporation. As tissue area is a natural limitation of autografting, mechanical strain was implemented to expand the periosteum. Freshly harvested, porcine periosteum was strained at 5 and 10% per day for 10 days with non-strained and free-floating samples serving as controls. Total tissue size, viability and histologic examination revealed that strain increased area to a maximum of 1.6-fold in the 10% daily strain. No change in tissue anatomy or viability via MTT or Ki67 staining and quantification was observed among groups. The osteogenic potential of the mechanical expanded periosteum was then examined in vivo. Human cancellous allografts were wrapped with 10% per day strained, fresh, free-floating, or no porcine periosteum and implanted subcutaneously into female, athymic mice. Tissue was collected at 8- and 16-weeks. Gene expression analysis revealed a significant increase in alkaline phosphatase and osteocalcin in the fresh periosteum group at 8-weeks post implantation compared to all other groups. Values among all groups were similar at week 16. Additionally, histological assessment with H&E and Masson-Goldner Trichrome staining showed that all periosteal groups outperformed the non-periosteal allograft, with fresh periosteum demonstrating the highest levels of new tissue mineralization at the periosteum-bone interface. Overall, mechanical expansion of the periosteum can provide increased area for segmental healing via autograft strategies, though further studies are needed to explore culture methodology to optimize osteogenic potential.

Fig 1. Harvesting of periosteum from the tibia of red Duroc pigs using a periosteal elevator.

Periosteum was cut into 2 x 4 cm strips and loaded into custom made 316L stainless steel strain devices with long-axis edges constrained in the lateral direction using sutures.

Fig 2. Ex vivo expansion of periosteal tissue is not linear with total applied strain.

A) Photographs of periosteum at day 0 and after 10 days of culture. Note the decrease in periosteal strip width in the 10% group. B) Quantification of periosteal area normalized to day 0.

Fig 3. Anatomy of periosteal tissue prior to and following in vitro culture.

H&E stained sections of fresh porcine periosteal tissue and periosteum after 10 days in culture. Scale bar = 100 μm.

Fig 4. Ex vivo expansion does not significantly alter cell proliferation or metabolism.

A) Quantification of Ki67⁺ cells per field of view as a function of culture condition. B) MTT cellular metabolism assay performed on punch biopsies collected from periosteum on culture day 10.

Fig 5. Mineralization in bone allograft following subcutaneous implantation.

Masson-Goldner staining of undecalcified sections from allograft only, allograft wrapped with fresh periosteum and allograft wrapped with mechanically expanded periosteum 16 weeks post-implantation.

Fig 6. Expression of genes related to bone regeneration in allograft alone or wrapped with periosteum.

Normalized gene expression for decorin, osteoprotegerin, alkaline phosphatase, and osteocalcin from samples collected at 8 and 16 weeks post-implantation.