Hydroxypropylmethyl cellulose (HPMC) crosslinked keratin/hydroxyapatite (HA) scaffold fabrication, characterization and in vitro biocompatibility assessment as a bone graft for alveolar bone regenerat

Wool derived keratin has garnered significant advancements in the field of biomaterials for hard tissue regeneration. The main limitation of keratin-based biomaterials for bone tissue engineering is their fragile nature. This paper proposes the development of a novel hydroxypropyl methylcellulose (HPMC) crosslinked keratin scaffold, containing hydroxyapatite as a major inorganic component by freeze drying technique for alveolar bone regeneration. The prepared keratin/hydroxyapatite/HPMC (K/HA/HPMC) scaffold was characterized to study its chemical, physical, and mechanical properties by Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDX), X-Ray diffractometric (XRD) analysis. The SEM images of the scaffolds showed highly porous interconnected architecture with average pore size of 108.36 ± 22.56 while microcomputed tomographic analysis measured total porosity as 79.65 %±. Energy dispersive X-ray spectroscopic (EDX) analysis confirmed that inorganic component of scaffold was mainly composed of calcium and phosphorous ions having Ca/P molar ration of 1.6. The maximum compressive strength was found to be in the range of 0.841 ± 0.37 MPa. Furthermore, the K/HA/HPMC scaffold was structurally stable and weight loss of about 26% was observed when soaked in phosphate buffered solution (PBS) for 28 days. In vitro biocompatibility testing showed that K/HA/HPMC scaffold was cytocompatible and supported the attachment, proliferation of osteoblast (Saos-2) cells. Thus, the development of a non-toxic chemical cross-linking system with HPMC was investigated to fabricate K/HA/HPMC scaffold and our results showed great potential of these scaffolds to regenerate alveolar bone due to their structural similarity and excellent in vitro biocompatibility.

1. Introduction

Alveolar bone which provides support to dentition is a dynamic structure undergoing continuous remodelling according to functional needs. There are numbers of local and systemic factors which results in progressive loss of this functional bone [1]. Most common clinical problem after tooth loss is the resorption of alveolar bone, especially in the anterior aesthetically important region. The application of dental implants to restore the lost teeth has now emerged as a very popular treatment modality due to overall procedure predictability, low failure rate, as well as its durability [2, 3, 4]. For the successful placement of titatnium screw implants, specific dimensions of alveolar ridge is crucial to allow greater available surface area for osseointegration [5]. Clinically the placement of short or narrow dental implants in resorbed alveolar bone regions is commonly practised, to achieve desirable prosthetic results. However, research in implant dentistry has reported lower success rates due to reduced implant to bone contact surface area to provide required anchorage and stability [6, 7] Thus, the role of tissue engineers becomes very important to developed materials or techniques to promote bone regeneration with predictable outcomes.

Bone grafting is extensively used in the field of orthopaedics to treat and reconstruct the bony defects. Currently, allografts and autografts are the most commonly used bone grafting procedure, but these often present some major drawbacks such as immunogenic reactions, morbidity of donor site, poor quality of donor bone due to underlying bone diseases etc. Thus, due to all these procedure related complexities, there is dire demand at both scientific and commercial level to develop more efficient treatment options that surpass the need of these allografts/autografts for bone regeneration. In last few decades, the field of tissue engineering has offered many advancements at nano level for hard tissue regeneration [8, 9, 10, 11]. Recently, fabrication of three dimensional, biocompatible, biodegradable scaffolds for bone regeneration has opened new perspective for research in this field. Freeze drying, 3-D printing, SBF immersion technique, cryogelation method, electrospinning are some of the extensively used procedures in the preparation of artificial hybrid matrices for treatment of damaged or diseased bony defects [12, 13, 14, 15, 16]. In our current study we report the preparation of K/HA/HPMC porous composite scaffold by using freeze drying technique. This technique also known as lyophilization, or ice templating has been demonstrated as a promising method for various tissue engineering applications [17, 18, 19]. With this technology 3-dimmensional scaffolds could be designed having more than 90% porosity and pore diameter in the range of 20–400μm [19, 20]. The porosity and shape of scaffolds can be managed by altering certain factors of the process, such as solution and instrumental parameters, which results in improved biological and mechanical properties of the scaffolds [20]. This process is studied on wide range of materials such as ceramics, organic – soluble polymer and water-soluble polymer [21, 22, 23, 24]. Freeze drying offers several advantages over other commonly used methods. Unlike electrospinning, water is used as solvent instead of toxic chemicals which makes overall process green and environment friendly as there are no chances of toxicity due to chemical residues in the prepared scaffolds [21, 25]. Furthermore, this method can be combined with other techniques such as gel casting, salt leaching, liquid dispensing for improved final properties of scaffolds [26, 27, 28, 29]. Thus, some of the practical limitations associated with conventional electrospinning technique such as insufficient mechanical strength for application in load bearing areas, possible toxicity of cross linkers or solvents, poor cellular infiltration can be overcome by freeze drying technology [25, 30, 31]. Another advantage associated with freeze drying is safe processing of heat sensitive drugs, growth factors or proteins as no heat is involved during the process.

During the past few years, there has been extensive research on keratin at both macro and nano scale as a potential material for various biomedical applications [8]. The distinct properties of this natural polymer such as intrinsic biological activity, mechanical durability, good biocompatibility results in the wide application of keratin in the field of modern regenerative medicine [32, 33]. Hair, wool, nails, feathers are some of the rich sources of this autogenous protein [8]. Natural bone is a composite structure mainly composed of inorganic biomineral (hydroxyapatite) which comprises of 60–70 % of bone, therefore, application of pure synthetic hydroxyapatite as a bone graft substitutes has been investigated extensively in number of in vitro and in vivo studies [34, 35, 36]. It was observed that it has osteogenic potential without causing any inflammatory response or toxicity. However, due to its brittle nature and poor strength these pure hydroxyapatite are not ideal substituted material for bone regeneration in load bearing areas [10]. To closely emulate the architecture of natural bone many researchers proposed different strategies to design porous scaffolds based on polymers and hydroxyapatite [32, 37]. Levingstone et al. designed a layered composite to closely mimic the bone density which gradually increases from inside out. However, this layered scaffold failed to achieve biomimetic function fully [37]. In the past, mixing of hydroxyapatite and keratin has proven to be a successful approach towards designing hybrid biomaterials having improved mechanical strength, excellent biocompatibility and desirable porosity [18, 38, 39]. Tachibana et al. proposed the immersion of keratin sponges in buffer solution containing calcium and phosphate ions. Though these composite sponges exhibited initial nucleation sites of the calcium phosphate, but scaffolds prepared by this technique lacked the continuous interconnected porous structure along with poor mechanical strength [39]. Our group previously worked on two techniques to produce porous keratin/HA scaffolds. These methods mainly involve mixing of ice microparticles to keratin/HA aqueous suspension and compression moulding. The results of in vivo implantation in animals showed good biocompatibility of these scaffolds. However, high density of these keratin/HA composite sponges and complex fabrication technique might limit their wide application in bone tissue engineering [38, 40].

Recently, cross-linking emerged as a promising strategy to design novel tissue engineered scaffolds with improved biomechanical properties specific to their application [41]. A cross-linking agent forms a chemical or physical bond to connect the polymeric chain functional groups through supramolecular bonding or covalent interactions [42]. Previously various cross-linking techniques have been utilized by researchers to modify the mechanical, biological and degradation properties of the fabricated scaffolds such as chemical crosslinking by glutaraldehyde or physical crosslinking by using dry heating treatment (DHT) etc [8, 43, 44]. Kim et al. cross-linked keratin/chitosan scaffolds with glutaraldehyde to prepare skin grafts [45]. Similarly, Li Chen et al. designed biomimetic porous collagen/HA scaffold for bone tissue engineering using glutaraldehyde as a cross-linking agent [17]. However, the presence of free aldehyde groups, when glutaraldehyde used as a cross-linker can cause cell toxicity and inflammatory reactions in the body. The glutaraldehyde cross-linked scaffolds require additional washing with amino acid or free amine groups solution to remove free aldehyde groups and to improve their biocompatibility [46]. . Therefore, there is a dire demand for exploring new technologies involving nontoxic, green chemicals for crosslinking of natural polymers.

Our current project is a part of an ongoing effort to design a composite scaffold closely mimicking the porous architecture of trabecular part of human alveolar bone using green, non-toxic cross linker to further improve the degradation, mechanical and biological properties of the scaffold. In this project, keratin was extracted using a patented technique and hydroxypropyl methyl cellulose (HPMC) was used as a cross-linking agent [47]. HPMC crosslinked, highly porous keratin scaffolds containing nanocrystalline hydroxyapatite as major inorganic component were fabricated by using a freeze-drying technique. HPMC is a hydrophilic, biodegradable polymer which is approved by United States Food and Drug Administration (FDA) for use in controlled release formulations [48]. The presence of HPMC provides additional sites of interaction for keratin and hydroxyapatite particles and thus aid in the formation of highly porous anisotropic structure. Previously, several studies showed the application of HPMC as a plasticizing agent to fill bone defects or to improve the mechanical properties of bone cements [49, 50, 51]. Ather et al. reported the preparation and characterization of highly porous chitosan/HA scaffolds using HPMC as a gelation agent [52]. Furthermore, another study reported the preparation of HPMC cross-linked chitosan scaffolds as a potential matrices for regeneration of alveolar bone [48]. As per our knowledge there is no study conducted so far to explore the application of HPMC as cross-linking agent for developing keratin derived scaffolds for bone regeneration. Therefore, the aim of our current investigation is to design interconnected, porous keratin/hydroxyapatite scaffolds utilising HPMC as a cross linker. After fabrication, the structure of the scaffold was investigated with using Scanning electron microscopy followed by Fourier transform infrared spectroscopy (FTIR), microcomputed tomography (u-CT) imaging, energy dispersive X-ray spectroscopy (EDX) and X-ray diffractometer (XRD). The degradation behaviour and in vitro biological activity of the scaffold was also investigated. The biocompatibility of the construct was studied by using human Saos-2 cell lines by cell viability and cell proliferation assays.