Elsevier

The Spine Journal

Volume 18, Issue 5, May 2018, Pages 818-830
The Spine Journal

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

https://doi.org/10.1016/j.spinee.2017.12.002Get rights and content

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.

Introduction

Spine fusion surgery is commonly performed to treat disc degenerative diseases affecting the lumbar and cervical spine. Traditionally, bone grafts taken from a patient's hip (autogenous grafts) are the most reliable means of achieving successful spinal fusion. However, such efforts are limited by insufficient supply, considerable donor site morbidity, chronic pain, and visceral hernia [1]. Furthermore, variation in the quality of the harvestable bone among patients is known to play a significant role in the inconsistent clinical outcomes observed among patients [2]. The development of synthetic scaffolds as alternatives to autogenous bone grafting has remained a key objective [3], [4], [5], [6].

In general, successful interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage that is filled with graft material and placed into the degenerated disc space. The type of bone graft alternative used to fill the fusion cage is typically chosen without regard for its mechanical performance. Instead, mechanical stability at the fusion site is reliant on fixation hardware combined with a selected cage. We reasoned that scaffolds used for spinal fusion not only should support the formation of new bone but also should be able to contribute to the mechanical stability at the fusion site rather than solely rely on the fixation hardware and cage. Therefore, such scaffolds should more closely match the mechanical properties of vertebral cancellous bone (stiffness=compressive modulus of 50–1,000 MPa; strength=compressive yield of 1–12 MPa) [7], [8], [9]. Polymeric alternatives to bone graft, like poly(ε-caprolactone) (PCL), have been used extensively to fabricate biomedical scaffolds [10], but their use is mostly limited to non–load-bearing sites because of their poor mechanical properties (stiffness of 2.7 MPa and strength of 1.5 MPa) [11], [12], [13], [14]. Improvements have been achieved (stiffness of 9.3 MPa and strength of 3.1 MPa) through the incorporation of β-tricalcium phosphate (β-TCP) into PCL scaffolds (PCL-TCP), albeit low interfacial bonding between β-TCP and PCL is known to weaken such constructs [7]. The addition of β-TCP also improves the osteoconductivity of scaffolds by introducing calcium and phosphate ions that more closely mimic the natural bone microenvironment [15]. Our group recently demonstrated that the addition of 3-glycidoxypropyl trimethoxysilane (GPTMS) greatly enhanced interfacial bonding between β-TCP and PCL [13]. The resultant PCL-GPTMS-TCP (silane-modified PCL-TCP [PCL-siTCP]) scaffolds are up to sixfold stronger than conventional PCL-TCP scaffolds, with a stiffness of 82.6 MPa and a strength of 4.0 MPa [13]. Such improved mechanical properties are much more closely aligned to those of cancellous bone.

To further enhance the biomimicry of PCL-siTCP scaffolds, we chose to incorporate a key regulatory element of the extracellular matrix (ECM), the glycosaminoglycan, heparan sulfate (HS) [16], [17]. Heparan sulfate is found both on cell surfaces [18] and throughout the bone ECM [16], [17]. There is increasing evidence showing that HS helps to regulate skeletal repair and regeneration, acting to modulate the activity of key reparative factors, particularly bone morphogenetic protein-2 (BMP2) [18], [19], VEGF165 [20], FGF2 [21], and TGF-β [22], [23], among others [19], [24]. Indeed, we have shown that the expression of HS proteoglycans during bone healing is closely aligned to the progression of new bone formation, suggesting that HS is temporally involved in the bone-healing process [25]. We have also shown that HS-functionalized materials act to bind, stabilize, and prolong the activity of growth factors, including BMP2 [19], [24], [26]. Also, trisulfated monosaccharides of HS can be functionalized to supramolecular nanostructures that bind and enhance the activity of BMP2 [27], highlighting the utility of these sugars for orthopedic purposes. Moreover, such an HS-coating strategy presents a unique opportunity to prepare novel surfaces tuned to bind and present growth factors in a more physiologically relevant manner. This technique then provides an opportunity to greatly lower the efficacious dose of osteoinductive factors like BMP2. We have developed an HS variant, HS3, that demonstrates an affinity for heparin-binding proteins such as BMP2 [28]. We have shown that collagen (Col)/HS3 matrices [28] and β-TCP/carboxymethyl cellulose/HS3 combination devices [29] enhance bone tissue repair by sequestering and protecting endogenous factors like BMP2 at sites of bone fracture. This finding highlights the importance of tissue-resident glycosaminoglycans found in particular microenvironments and the diverse roles they play in helping orchestrate growth factor-mediated tissue responses.

We, therefore, sought to combine the synthetic bone graft alternative PCL-siTCP (whose mechanical and osteoconductive properties closely match cancellous bone) with Col and the HS variant HS3 that has been shown to prolong the bioactivity of BMP2. The ability of this combination device (PCL-siTCP/Col/HS3) to support progenitor cell proliferation and BMP2-mediated osteogenic differentiation was determined. As a prelude to future studies in spinal fusion models, the in vivo bone-forming ability of the combination device was assessed using an ectopic bone model that exploited the hamstring muscles of rats. In fact, this model was chosen because initial bone induction studies of BMP/hydroxyapatite/Col implants were performed in a dorsal muscle model [30]. Furthermore, because the success of spinal fusion procedures is contingent on multiple host and graft characteristics [31] that are conducive to bone formation, a detailed assessment of implant osteoconductivity and osteoinductivity should be performed. Failure to do so could adversely affect the future clinical application of the technology. An evaluation of bone formation at ectopic sites provides such validating evidence and helps to provide an evaluation of uncontrolled bone formation that has been associated with the use of high-dose BMP2.

The data from both in vitro and in vivo studies suggest that major improvements in the mechanical performance of scaffolds being developed for spinal fusion procedures can be achieved without compromising biological performance. Moreover, adding ECM-mimicking glycosaminoglycans, like HS, can be used to further drive the performance of these scaffolds for the delivery of osteogenic factors such as BMP2.

Section snippets

Materials

All reagents and chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. Recombinant human BMP2 was from Medtronic INFUSE Sofamor Danek (Memphis, TN, USA). The enzyme-linked immunosorbent assay (ELISA) kit for BMP2 was purchased from R&D Systems (Minneapolis, MN, USA). Heparan sulfate (HS3) was prepared as previously described [28]. Silanated β-TCP and PCL-siTCP scaffolds were also fabricated as described previously [32].

Scaffold preparation

From scaffold sheets of PCL-siTCP,

Distribution of BMP2 and HS3 within PCL-siTCP/Col scaffolds

Previous studies have shown that silanized PCL scaffolds containing ceramic TCP particles (PCL-siTCP) exhibit improved interfacial bonding between the polymer matrix and ceramic particles [32], thus increasing the overall compressive strength of the scaffold by 600% as compared with conventional PCL-TCP scaffolds. The highly porous nature of silanized PCL-TCP/Col scaffolds makes them amenable to functionalization with BMP2, either alone or complexed with glycosaminoglycans [33]. Scanning

Discussion

Mechanical stability at the site and maintenance of height are both critical to the success of spinal fusion procedures [44]. For this reason, the current standard of care for interbody fusion necessitates external instrumentation (plates, rods, and screws) and the placement of a fusion cage with mechanical properties sufficient to withstand the high loading environment of the spine. The fusion cage is typically filled with bone graft to stimulate fusion between adjacent spinal segments.

Conclusions

The results of the present study demonstrate an increased consistency in the osteostimulatory performance of PCL-siTCP/Col/HS3 scaffolds codelivering BMP2 compared with scaffolds without the bone matrix mimicking glycosaminoglycan HS3. Moreover, tissue filling of the pores of the PCL-siTCP/Col/HS3/BMP2 scaffolds is considerably more mineralized, with signs of an increasing number and thickness of bony structures. This finding suggests that such PCL-siTCP/Col/HS3 devices offer a promising

Acknowledgments

The authors thank the funding support from the Joint Council Office (JCO) of Singapore's Agency for Science, Technology and Research (A*STAR) (Grant JCOAG04_FG04_2009) and the Institute of Medical Biology (IMB), A*STAR and the Institute of Materials Research and Engineering, A*STAR. Authors are also thankful to Prof. Paulo Bartolo, Polytechnic Institute of Leiria, Portugal, for fabricating PCL-siTCP scaffolds.

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    FDA device/drug status: Not applicable.

    Author disclosures: GB: Nothing to disclose. AKE: Nothing to disclose. BR: Nothing to disclose. SAA: Nothing to disclose. TCT: Nothing to disclose. BQL: Nothing to disclose. AC: Nothing to disclose. TH: Nothing to disclose. TL: Nothing to disclose. MTA: Nothing to disclose. AJvW: Nothing to disclose. JG: Nothing to disclose. VN: Stock Ownership: SMC Biotechnology Inc. (B), outside the submitted work. KB: Nothing to disclose. WB: Nothing to disclose. LX: Nothing to disclose. IG: Nothing to disclose. H-KW: Royalties: SpineGuard (B); Consulting: SpineGuard (B), outside the submitted work. SMC: Stock Ownership: SMC Biotechnology Inc. (B), outside the submitted work.

    The disclosure key can be found on the Table of Contents and at www.TheSpineJournalOnline.com.

    The work reported in this publication was wholly supported by public host-institution funding. No support in any form was received from an industry sponsored research program or industry source. We therefore report no conflict of interest.

    1

    Present address: Science and Math Cluster, Singapore University of Technology and Design (SUTD), 8 Somapah Rd, Singapore 487372.

    2

    Present address: Centre for Research in Medical Devices (CÚRAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland.

    3

    Present address: Department of Biomedical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka-1205, Bangladesh.

    4

    Present address: School of Engineering, Deakin University, CADET Building, Waurn Ponds Campus, Victoria, Australia 3216.

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