Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications

Gajadhar Bhakta, Andrew K. Ekaputra, Bina Rai, Sunny A. Abbah, Tuan Chun Tan, Q. le Bach, Anindita Chatterjea, Tao Hu, Tingxuan Lin, M. Tarik Arafat, Andre J. van Wijnen, James Goh, Victor Nurcombe, Kishore Bhakoo, William R. Birch, Li Xu, Ian Gibson, Hee Kit Wong, Simon Cool (Corresponding Author)

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Scopus)

Abstract

Abstract
Background Context
Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site.

Purpose
We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device.

Study Design/Setting
A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds.

Materials and Methods
Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120–150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology.

Results
Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants.

Conclusions
The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.
Original languageEnglish
Pages (from-to)818-830
JournalSpine journal
Volume18
Issue number5
DOIs
Publication statusPublished - 2018
Externally publishedYes

Fingerprint

Spinal Fusion
Heparitin Sulfate
Collagen
Osteogenesis
Osteocalcin
Bone and Bones
Equipment and Supplies
Cell Differentiation
Histology
polycaprolactone
tricalcium phosphate
Stem Cells
Cell Proliferation
Transplants
Silanes
X-Ray Microtomography
Bone Morphogenetic Protein 2
Messenger RNA
Bone Matrix
Glycosaminoglycans

Cite this

Bhakta, Gajadhar ; Ekaputra, Andrew K. ; Rai, Bina ; Abbah, Sunny A. ; Tan, Tuan Chun ; le Bach, Q. ; Chatterjea, Anindita ; Hu, Tao ; Lin, Tingxuan ; Arafat, M. Tarik ; van Wijnen, Andre J. ; Goh, James ; Nurcombe, Victor ; Bhakoo, Kishore ; Birch, William R. ; Xu, Li ; Gibson, Ian ; Wong, Hee Kit ; Cool, Simon. / Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications. In: Spine journal. 2018 ; Vol. 18, No. 5. pp. 818-830.
@article{aa9af07663994c04854584fcb2382938,
title = "Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications",
abstract = "AbstractBackground ContextInterbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site.PurposeWe recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device.Study Design/SettingA combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds.Materials and MethodsFreshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120–150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology.ResultsProgenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants.ConclusionsThe combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.",
author = "Gajadhar Bhakta and Ekaputra, {Andrew K.} and Bina Rai and Abbah, {Sunny A.} and Tan, {Tuan Chun} and {le Bach}, Q. and Anindita Chatterjea and Tao Hu and Tingxuan Lin and Arafat, {M. Tarik} and {van Wijnen}, {Andre J.} and James Goh and Victor Nurcombe and Kishore Bhakoo and Birch, {William R.} and Li Xu and Ian Gibson and Wong, {Hee Kit} and Simon Cool",
year = "2018",
doi = "10.1016/j.spinee.2017.12.002",
language = "English",
volume = "18",
pages = "818--830",
journal = "Spine journal",
issn = "1529-9430",
publisher = "Elsevier",
number = "5",

}

Bhakta, G, Ekaputra, AK, Rai, B, Abbah, SA, Tan, TC, le Bach, Q, Chatterjea, A, Hu, T, Lin, T, Arafat, MT, van Wijnen, AJ, Goh, J, Nurcombe, V, Bhakoo, K, Birch, WR, Xu, L, Gibson, I, Wong, HK & Cool, S 2018, 'Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications' Spine journal, vol. 18, no. 5, pp. 818-830. https://doi.org/10.1016/j.spinee.2017.12.002

Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications. / Bhakta, Gajadhar; Ekaputra, Andrew K.; Rai, Bina; Abbah, Sunny A.; Tan, Tuan Chun; le Bach, Q.; Chatterjea, Anindita; Hu, Tao; Lin, Tingxuan; Arafat, M. Tarik; van Wijnen, Andre J.; Goh, James; Nurcombe, Victor; Bhakoo, Kishore; Birch, William R.; Xu, Li; Gibson, Ian ; Wong, Hee Kit; Cool, Simon (Corresponding Author).

In: Spine journal, Vol. 18, No. 5, 2018, p. 818-830.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications

AU - Bhakta, Gajadhar

AU - Ekaputra, Andrew K.

AU - Rai, Bina

AU - Abbah, Sunny A.

AU - Tan, Tuan Chun

AU - le Bach, Q.

AU - Chatterjea, Anindita

AU - Hu, Tao

AU - Lin, Tingxuan

AU - Arafat, M. Tarik

AU - van Wijnen, Andre J.

AU - Goh, James

AU - Nurcombe, Victor

AU - Bhakoo, Kishore

AU - Birch, William R.

AU - Xu, Li

AU - Gibson, Ian

AU - Wong, Hee Kit

AU - Cool, Simon

PY - 2018

Y1 - 2018

N2 - AbstractBackground ContextInterbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site.PurposeWe recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device.Study Design/SettingA combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds.Materials and MethodsFreshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120–150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology.ResultsProgenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants.ConclusionsThe combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.

AB - AbstractBackground ContextInterbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site.PurposeWe recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device.Study Design/SettingA combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds.Materials and MethodsFreshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120–150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology.ResultsProgenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants.ConclusionsThe combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.

U2 - 10.1016/j.spinee.2017.12.002

DO - 10.1016/j.spinee.2017.12.002

M3 - Article

VL - 18

SP - 818

EP - 830

JO - Spine journal

JF - Spine journal

SN - 1529-9430

IS - 5

ER -