The character of gene expression of human periosteum used to form new tissue in allograft bone

Abstract

Of more than 2 million segmental bone defects repaired annually with bone autografts and allografts, 15–40% fail. Improving healing rates may be approached with tissue engineering and use of periosteum overlying an allograft. The present study documents gene expression in human periosteum–allograft constructs compared to allografts alone. Strips of human cadaveric periosteum (26 years, f, distal femur) were sutured about sterilized human femoral cortical strut bone allograft (54 years, m) segments. After construct incubation (M199 supplemented medium) for 8 d, constructs and allografts alone were implanted in nude mice. At 10 and 20 weeks, constructs (N = 4, each group) and allografts (N = 2, each group) were retrieved and placed in RNAlater for quantitative PCR to determine expression of human- and murine-specific genes relevant to remodeling. Specimens were frozen-ground to powders and RNA was extracted, purified, reverse-transcribed, and amplified. Ribosomal protein (P0) was used to normalize sample quantities. Fold change plots were generated following statistical analyses comparing 20- to 10-week gene expression data. Allografts alone yielded no human-specific gene expression. Notable fold changes of human-specific alkaline phosphatase, bone sialoprotein, type I collagen, decorin, RANKL, RANK, cathepsin K, and osteocalcin in 20-week compared to 10-week specimens were found. Murine-specific expression of genes indicative of host mouse vascularization (RANK, type I collagen) was detected in both allograft alone and periosteum–allograft samples. Gene data confirm viable periosteum in constructs after 20 weeks. Relatively higher fold-change values of RANK, RANKL and cathepsin K indicate activities of osteoclast precursors, osteoclasts and osteoblasts involved in allograft remodeling during implantation. All additional genes of interest indicate osteoblast activity in new bone matrix formation. Gene data are directly correlated with previous and present histology work. The results of this study suggest that further investigations could help to establish whether autologous periosteum–allograft constructs could be used for the repair of bone defects.

• Allograft bone
• fracture healing
• gene expression
• periosteum
• tissue engineering

Acknowledgments

The authors thank the Sarcoma Research Fund (Ohio State University) for funding support, the Gift of Hope Organ & Tissue Donor Network (Elmhurst, IL) and Dr. Susan Chubinskaya (Rush University, Chicago) for human donor tissue, the Musculoskeletal Transplant Foundation (Jessup, PA) for donation of allograft bone, Ms. Mary Beth Wade (University of Akron) and Mr. Mark Shasti (Northeast Ohio Medical University) for assistance with implants, and the Northeast Ohio Medical University Comparative Medicine Unit for animal care and maintenance. The authors are especially grateful to the families of donors for access to tissues.