Basic ScienceFabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications
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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Cited by (12)
Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic
2023, Biomaterials AdvancesBiological role of heparan sulfate in osteogenesis: A review
2021, Carbohydrate PolymersCitation Excerpt :Meanwhile, HSPGs are also considered as the essential reagents in the process of bone development. In recent years, with the development of bone tissue engineering, some scholars gradually used HS as a bone tissue engineering material (Bhakta et al., 2018; Le et al., 2019). Some scholars even introduced HS into 3D printing tissue engineering materials to further expand the applications of HS in osteogenesis (Liu, Wang, et al., 2020; Liu, Xu, et al., 2020).
A biomimetic collagen-bone granule-heparan sulfate combination scaffold for BMP2 delivery
2021, GeneCitation Excerpt :In general accordance with the concept of biomimicry, we previously developed an HS glycosaminoglycan variant with increased affinity for BMP2 (HS3) designed to mimic such sugars from the extracellular niche of bone tissue (Murali et al., 2013). When functionalized to bioscaffolds, we show that HS3 enhances the endogenous bone healing process (Murali et al., 2013, 2009; Ling et al., 2006, 2010; Manton et al., 2006; Jackson et al., 2007; Bramono et al., 2012; Rai, 2015; Bhakta, 2018; Le, 2019), confirming the critical role of HS in modulating growth factor-mediated events during both development and repair (Cool and Nurcombe, 2006; Song et al., 2006). Here we report on the development of a collagen/ bone granule (Intergraft™)/HS3 (Col/In/HS3) scaffold intended to deliver BMP2 with sustained release.
Three-dimensional printing in spine surgery: a review of current applications
2020, Spine JournalCitation Excerpt :Biodegradable fusion cages have been constructed with medical grade PCL and infused with rh-BMP2. Preclinical in vivo and in vitro animal studies have shown that PCL fusion cages may contribute both to the formation of new bone and to mechanical stability at the fusion site [80–82]. Additionally, Knutsen et al. (2015) [83] designed a bioresorbable cervical fusion cage from PCL, which can theoretically withstand a considerable load when combined with supplemental fixation.
A polycaprolactone-β-tricalcium phosphate–heparan sulphate device for cranioplasty
2019, Journal of Cranio-Maxillofacial SurgeryCitation Excerpt :In these studies, we exemplified the ability of HS3 to support endogenous bone healing cascades when combined with a range of clinically-approved materials, including collagen (Murali et al., 2013) and β-TCP/carboxymethyl cellulose (CMC) (Rai et al., 2015) devices. We have since developed an HS3-silane-modified PCL–TCP/collagen device as a scaffold for the delivery of BMP-2 for spinal fusion applications (Bhakta et al., 2017). Furthermore, HS-like materials have also enhanced tissue regeneration when functionalized to materials (Albo et al., 1996; Lafont et al., 1998; Escartin et al., 2003; Lee et al., 2017), providing additional support for the use of glycosaminoglycan-based strategies in regenerative medicine.
Clinical applications of bioactive materials
2019, Materials for Biomedical Engineering: Bioactive Materials, Properties, and Applications
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.