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, Bach Quang Le, Anindita Chatterjea, Tao Hu, Tingxuan Lin, M. Tarik Arafat, Andre J van Wijnen, James Goh, Victor Nurcombe, Kishore Bhakoo, William Birch, Li Xu, Ian Gibson, Hee Kit Wong, Simon M. Cool

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 (US)
JournalSpine Journal
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

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

Keywords

  • Bone morphogenetic protein-2
  • Collagen
  • Ectopic bone
  • Glycosaminoglycans
  • PCL
  • Spinal fusion

ASJC Scopus subject areas

  • Surgery
  • Clinical Neurology

Cite this

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 Quang; Chatterjea, Anindita; Hu, Tao; Lin, Tingxuan; Arafat, M. Tarik; van Wijnen, Andre J; Goh, James; Nurcombe, Victor; Bhakoo, Kishore; Birch, William; Xu, Li; Gibson, Ian; Wong, Hee Kit; Cool, Simon M.

In: Spine Journal, 01.01.2018.

Research output: Contribution to journalArticle

Bhakta, G, Ekaputra, AK, Rai, B, Abbah, SA, Tan, TC, Le, BQ, Chatterjea, A, Hu, T, Lin, T, Arafat, MT, van Wijnen, AJ, Goh, J, Nurcombe, V, Bhakoo, K, Birch, W, Xu, L, Gibson, I, Wong, HK & Cool, SM 2018, 'Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications', Spine Journal. https://doi.org/10.1016/j.spinee.2017.12.002
Bhakta, Gajadhar ; Ekaputra, Andrew K. ; Rai, Bina ; Abbah, Sunny A. ; Tan, Tuan Chun ; Le, Bach Quang ; Chatterjea, Anindita ; Hu, Tao ; Lin, Tingxuan ; Arafat, M. Tarik ; van Wijnen, Andre J ; Goh, James ; Nurcombe, Victor ; Bhakoo, Kishore ; Birch, William ; Xu, Li ; Gibson, Ian ; Wong, Hee Kit ; Cool, Simon M. / Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications. In: Spine Journal. 2018.
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title = "Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications",
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.",
keywords = "Bone morphogenetic protein-2, Collagen, Ectopic bone, Glycosaminoglycans, PCL, Spinal fusion",
author = "Gajadhar Bhakta and Ekaputra, {Andrew K.} and Bina Rai and Abbah, {Sunny A.} and Tan, {Tuan Chun} and Le, {Bach Quang} 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 William Birch and Li Xu and Ian Gibson and Wong, {Hee Kit} and Cool, {Simon M.}",
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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 Quang

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

AU - Xu, Li

AU - Gibson, Ian

AU - Wong, Hee Kit

AU - Cool, Simon M.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - Bone morphogenetic protein-2

KW - Collagen

KW - Ectopic bone

KW - Glycosaminoglycans

KW - PCL

KW - Spinal fusion

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