Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis

Category: Basic Sciences/Biologics; Ankle Introduction/Purpose: One of the most common procedures for treating end-stage osteoarthritis of the foot and ankle is arthrodesis [1]. However, a significant complication with arthrodesis is the development of non-union. Thus, identification of adjuvants to...

Full description

Bibliographic Details
Main Authors: Nicholas Huffman BS, Bereket Getachew, Jason T. Bariteau MD, Jay M. Patel PhD, Sreedhara Sangadala PhD
Format: Article
Language:English
Published: SAGE Publishing 2023-12-01
Series:Foot & Ankle Orthopaedics
Online Access:https://doi.org/10.1177/2473011423S00134
_version_ 1797375520130727936
author Nicholas Huffman BS
Bereket Getachew
Jason T. Bariteau MD
Jay M. Patel PhD
Sreedhara Sangadala PhD
author_facet Nicholas Huffman BS
Bereket Getachew
Jason T. Bariteau MD
Jay M. Patel PhD
Sreedhara Sangadala PhD
author_sort Nicholas Huffman BS
collection DOAJ
description Category: Basic Sciences/Biologics; Ankle Introduction/Purpose: One of the most common procedures for treating end-stage osteoarthritis of the foot and ankle is arthrodesis [1]. However, a significant complication with arthrodesis is the development of non-union. Thus, identification of adjuvants to enhance osteogenesis are paramount to fusion success. One possible molecule is the immunosuppressant FK506 (Tacrolimus). Previous research [2] demonstrated FK506 as a stand-alone small molecule capable of initiating osteogenesis and bone formation. The purpose of this study was to evaluate the osteogenic potential of FK506 alone and in combination with other bioactive factors to provide a foundation for future in vivo and clinical applications. Methods: Marrow-derived cells (MDCs) were isolated from juvenile bovine femoral condyles, to represent cells present at the ankle fusion site. MDCs (P1) were seeded at 50k cells per well (24-well plate). After 14 days, the wells were stained for calcium deposits using Alizarin Red S, and imaged with brightfield microscopy to visualize calcium deposits. Drug combinations of potentially osteogenic small molecules were tested (simvastatin, platelet derived growth factor [PDGF], tamoxifen, triiodothyronine). The highest alizarin red absorbance values were used to choose the best drug combination. These combinations were further evaluated for gene expression by RT-qPCR [Osteocalcin (OCN), Osteopontin (OPN), and Bone Sialoprotein (IBSP)]. To simulate a 3D osteogenic environment, cells were seeded into fibrin gels (to mimic the “fusion clot”), cultured for 14 days, and sectioned/stained with Alizarin red. Results: FK506 and PDGF produced the highest level of calcium staining compared to other bioactive factors, highlighted via heatmap of alizarin red absorbance values (Figure 1A). Compared to the control, FK506, PDGF, and the combination of both all resulted in enhanced alizarin red staining (Figure 1A/B). Overall, FK506 exhibited an increased expression across all genes. PDGF and the combination of FK506 and PDGF had a more varied expression within the genes queried (Figure 1C). Finally, in a fibrin gel system, variability was observed between gels of the same group. Displaying the best, middle, and worst sections give a comprehensive view of the explained variability. Across the sections, the FK506-PDGF combination showed the densest mineralization, highlighted by the dark maroon regions of the section (Figure 1D). Conclusion: This study confirms that FK506 demonstrates osteogenesis within marrow-derived cells (representative of a fusion site). The combination of FK506 to PDGF may enhance PDGF’s known healing potential within arthrodesis applications. This work provides the foundation for future clinical applications of FK506. Ongoing studies are focused on the effect of FK506, and potentially in combination with PDGF, in an rabbit ankle fusion model to evaluate efficacy and bone formation. The authors thank the American Orthopaedic Foot and Ankle Society (AOFAS) and Emory Department of Orthopaedics for their support and funding. References: [1] Park 2022. [2] Sangadala 2019
first_indexed 2024-03-08T19:25:38Z
format Article
id doaj.art-7bd29be15c254e269cb5f4f740a630f3
institution Directory Open Access Journal
issn 2473-0114
language English
last_indexed 2024-03-08T19:25:38Z
publishDate 2023-12-01
publisher SAGE Publishing
record_format Article
series Foot & Ankle Orthopaedics
spelling doaj.art-7bd29be15c254e269cb5f4f740a630f32023-12-26T09:06:13ZengSAGE PublishingFoot & Ankle Orthopaedics2473-01142023-12-01810.1177/2473011423S00134Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle ArthrodesisNicholas Huffman BSBereket GetachewJason T. Bariteau MDJay M. Patel PhDSreedhara Sangadala PhDCategory: Basic Sciences/Biologics; Ankle Introduction/Purpose: One of the most common procedures for treating end-stage osteoarthritis of the foot and ankle is arthrodesis [1]. However, a significant complication with arthrodesis is the development of non-union. Thus, identification of adjuvants to enhance osteogenesis are paramount to fusion success. One possible molecule is the immunosuppressant FK506 (Tacrolimus). Previous research [2] demonstrated FK506 as a stand-alone small molecule capable of initiating osteogenesis and bone formation. The purpose of this study was to evaluate the osteogenic potential of FK506 alone and in combination with other bioactive factors to provide a foundation for future in vivo and clinical applications. Methods: Marrow-derived cells (MDCs) were isolated from juvenile bovine femoral condyles, to represent cells present at the ankle fusion site. MDCs (P1) were seeded at 50k cells per well (24-well plate). After 14 days, the wells were stained for calcium deposits using Alizarin Red S, and imaged with brightfield microscopy to visualize calcium deposits. Drug combinations of potentially osteogenic small molecules were tested (simvastatin, platelet derived growth factor [PDGF], tamoxifen, triiodothyronine). The highest alizarin red absorbance values were used to choose the best drug combination. These combinations were further evaluated for gene expression by RT-qPCR [Osteocalcin (OCN), Osteopontin (OPN), and Bone Sialoprotein (IBSP)]. To simulate a 3D osteogenic environment, cells were seeded into fibrin gels (to mimic the “fusion clot”), cultured for 14 days, and sectioned/stained with Alizarin red. Results: FK506 and PDGF produced the highest level of calcium staining compared to other bioactive factors, highlighted via heatmap of alizarin red absorbance values (Figure 1A). Compared to the control, FK506, PDGF, and the combination of both all resulted in enhanced alizarin red staining (Figure 1A/B). Overall, FK506 exhibited an increased expression across all genes. PDGF and the combination of FK506 and PDGF had a more varied expression within the genes queried (Figure 1C). Finally, in a fibrin gel system, variability was observed between gels of the same group. Displaying the best, middle, and worst sections give a comprehensive view of the explained variability. Across the sections, the FK506-PDGF combination showed the densest mineralization, highlighted by the dark maroon regions of the section (Figure 1D). Conclusion: This study confirms that FK506 demonstrates osteogenesis within marrow-derived cells (representative of a fusion site). The combination of FK506 to PDGF may enhance PDGF’s known healing potential within arthrodesis applications. This work provides the foundation for future clinical applications of FK506. Ongoing studies are focused on the effect of FK506, and potentially in combination with PDGF, in an rabbit ankle fusion model to evaluate efficacy and bone formation. The authors thank the American Orthopaedic Foot and Ankle Society (AOFAS) and Emory Department of Orthopaedics for their support and funding. References: [1] Park 2022. [2] Sangadala 2019https://doi.org/10.1177/2473011423S00134
spellingShingle Nicholas Huffman BS
Bereket Getachew
Jason T. Bariteau MD
Jay M. Patel PhD
Sreedhara Sangadala PhD
Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
Foot & Ankle Orthopaedics
title Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
title_full Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
title_fullStr Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
title_full_unstemmed Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
title_short Combinatorial Small Molecule Enhancement of Osteogenesis for Ankle Arthrodesis
title_sort combinatorial small molecule enhancement of osteogenesis for ankle arthrodesis
url https://doi.org/10.1177/2473011423S00134
work_keys_str_mv AT nicholashuffmanbs combinatorialsmallmoleculeenhancementofosteogenesisforanklearthrodesis
AT bereketgetachew combinatorialsmallmoleculeenhancementofosteogenesisforanklearthrodesis
AT jasontbariteaumd combinatorialsmallmoleculeenhancementofosteogenesisforanklearthrodesis
AT jaympatelphd combinatorialsmallmoleculeenhancementofosteogenesisforanklearthrodesis
AT sreedharasangadalaphd combinatorialsmallmoleculeenhancementofosteogenesisforanklearthrodesis