A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone

Abstract

A novel immunohistochemistry (IHC) approach has been developed to label and localize osterix, a bone-specific transcription factor, within formalin-fixed, paraffin-embedded, tissue-engineered constructs uniquely containing synthetic polymers and human periosteal tissue. Generally, such specimens consisting in part of polymeric materials and mineral are particularly difficult for IHC identification of proteins. Samples here were fabricated from human periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds and harvested following 10 weeks of implantation in athymic mice. Heat-induced and protease-induced epitope retrieval methods from selected existing protocols were examined to identify osterix. All such protease-induced techniques were unsuccessful. Heat-induced retrieval gave positive results for osterix immunohistochemical staining in sodium citrate/EDTA/Tween 20 with high heat (120C) and pressure (~30 psi) for 10 min, but the heat and pressure levels resulted in tissue damage and section delamination from slides that limited protocol effectiveness. Heat-induced epitope retrieval led to other osterix-positive staining results achieved with minimal impact on structural integrity of the tissue and polymers in sodium citrate/EDTA/Tween 20 buffer at 60C and normal pressure (14.5 psi) for 72 hr. The latter approach identified osterix-positive cells by IHC within periosteal tissue, layers of electrospun PLLA nanofibers, and underlying PCL/PLLA scaffolds of the tissue-engineered constructs.

Silica/polycaprolactone nanofiber scaffold variants for human periosteal cell growth

“…By Day 21 in culture, the cells appeared to have migrated from the scaffold surface on which they were applied to populate more completely the respective mats. In this regard, seeded periosteal cells are capable of moving into and throughout the scaffolds on which they grow, a result also reported recently for osteoblasts migrating from human donor periosteum into and through a poly‐ l ‐lactic acid (PLLA) nanofiber layer overlying a PCL/PLLA scaffold …”

“…in bone nonunion and treatments such as distraction osteogenesis, 37 the presence of an intact periosteum extending across the involved bone segments is a singular necessity for healing. 3,4,36 In this context, periosteum favorably affects the formation of new bone and cartilage in tissue-engineered models of normal osteogenesis, 3,28,35 and these reports have served in part as the basis for the present investigation. Periosteum, however, is often insufficient in quantity to meet the requirement for healing and, as a consequence, nonunion and other difficult bone injuries present significant clinical challenges including major surgical procedures with only limited success.…”

“…The same four allograft–periosteum–nanoPCL scaffold constructs fixed and examined by H&E staining were studied by immunohistochemical staining for osterix, a definitive marker of bone cells . Osterix immunohistochemistry followed a protocol developed in this laboratory previously . Briefly, paraffin sections mounted on slides were deparaffinized and then rehydrated before additional surface decalcification was made in Immunocal (2 × 1 h; StatLab Medical Products).…”

“…[30][31][32][33][34] Osterix immunohistochemistry followed a protocol developed in this laboratory previously. 35 Briefly, paraffin sections mounted on slides were deparaffinized and then rehydrated before additional surface decalcification was made in Immunocal (2 × 1 h; StatLab Medical Products). Osterix was detected in sections by means of antibody reactivity with anti-osterix (Santa Cruz Biotechnologies, Dallas, TX) in a 1:50 dilution with Leica antibody diluent (Leica Biosystems, Inc., Buffalo Grove, IL), incubation at 4 C overnight and use with an Impress kit (Vector Laboratories, Burlingame, CA).…”

“…Osterix was detected in sections by means of antibody reactivity with anti-osterix (Santa Cruz Biotechnologies, Dallas, TX) in a 1:50 dilution with Leica antibody diluent (Leica Biosystems, Inc., Buffalo Grove, IL), incubation at 4 C overnight and use with an Impress kit (Vector Laboratories, Burlingame, CA). 35 Visualization of osterix was accomplished with 3,3 0 -diaminobenzidine (MaxTag DAB tablets; Rockland, Limerick, PA). Slides were counterstained with hematoxylin before being dehydrated, coverslipped, and documented by light microscopy as described above.…”