Abstract
Numerous biomaterials are used to fabricate bone scaffolds to repair the bones subjected to trauma. The scaffolds are fabricated with interconnected pores with 40–70% porosity to facilitate the entry of the cells that ensures rapid bone formation. In addition, the interconnected pores also serve as a channel for the exchange of nutrients and waste materials. Rapid prototyping techniques use the CAD model of the scaffold to be fabricated which facilitates fabrication of components with complex architecture easily. This research deals with the design, fabrication and analysis of porous scaffold models with different configurations. Apart from the conventional pore geometry like cubical, spherical shaped pores, their shifted arrangements were also considered for this study. The minimum pore size used for the study is 400 μm and the porosity ranges from 40–70%. Based on the results of finite element analysis, the best scaffold configuration is identified and was fabricated with different build orientation using Selective Laser Sintering (SLS) process with different mix of Polyamide and Hydroxyapatite. The fabricated test specimens were evaluated based on mechanical tests for its strength and in vitro studies with human osteosarcoma cell line for cell growth studies. The mechanical tests witnesses good physical properties than the earlier reported research. The suitability of the porous scaffolds for bone repair is also ensured using finite element analysis of a human femur bone under various physical activities.
Keywords
Introduction
Osteoporosis is a common disease with skeletal fragility, low bone mass and bones becomes very weak and more likely to fracture, in particular at the pelvis, hips, wrists and spine [1]. It has been reported that 1 in 3 women and 1 in 5 men around the world are at risk of an osteoporotic fracture. In fact, an osteoporotic fracture is estimated to occur every 3 seconds. It has been estimated about 9 million osteoporotic fractures worldwide in 2000 of which 1.4 million clinical vertebral fractures, 1.6 million were hip and 1.7 million forearm [2]. There were an estimated 700,000 vertebral compression factures each year which is one type of spinal fractures caused by osteoporosis. It has been reported by expert groups peg that in India alone, osteoporosis patients are approximately 26 million and this number will be projected to increase [3]. A study has also proved that among Indian women aged 30–60 years from low income groups, bone mineral density at all the skeletal sites were much lower than values reported from developed countries, with a high prevalence of osteopenia (52%) and osteoporosis (29%) thought to be due to inadequate nutrition [4]. The number of head and bone injuries among Indian population due to road traffic accidents is growing in an enormous rate, reported annually by the Transport Research Wing of the Ministry of Road Transport & Highways and National Crimes Records Bureau of Ministry of Home Affairs, Government of India. According to the World Health Organization, road traffic injuries are sixth leading cause of death in India with a greater share of hospitalization, deaths and disabilities in the young and middle-aged population [5]. Bone geometry i.e. bone shape and size, bone mineral density, microarchitecture, degree of mineralization are the properties that contribute to bone strength [6]. Fabrication of biomaterial into 3-D scaffold structures is the next vital step in the development of bone implants depending on bone injuries of individual patients and it is highly demanding among the Indian surgeons for treating those bone related defects. Under this present situation, fabrication of scaffolds by using 3-D printing such as Selective Laser Sintering could be the promising and viable alternative for treating bone related disorders.
Therefore, the need for reliable and economically feasible design, biomaterials, fabrications for healing fractures or treating musculoskeletal defects has increased in recent years. The perfect design, biomaterial, fabrication and implant for replacement of fractured or injured bone with combined features of biocompatibility and mechanical strength are still under the development not only in India, also throughout the world.
Investigation of scaffold for porous structured bone implant is shown in Fig. 1 is a recently emerging field in medicine and is involved in developing artificial bones like structure using materials like Tri Calcium Phosphate, polyetheretherketone, Hydroxyapatite, Polycaprolactone, poly(l-lactide) PLLA or Polyamide by incorporating pores in the scaffold [7–10]. All the available feasible methods focus on making scaffolds without pores incorporated in them, or by the use of grafting procedures, wherein, the porosity factor need not be addressed due to the innate capabilities that aid in pore formation. Natural bones are made up of calcium phosphate compounds and have three dimensional microscopic pore networks [11–13]. The usage of CAD software is also of a great backing in the development of the porous scaffolds. The capability of the human bones to fuse scaffolds has also encouraged its development. In this work, potential design, analysis of different structures, fabrication and characterization of fabricated materials are investigated for the perfect replacement of bone. The physical testing also witnesses an improved mechanical properties than that reported in the literature [14]. Then, in vitro cell culture studies were carried out on the specimens to predict the growth behaviour of live cells inside the scaffold.
Human femur with the implanted scaffold.
Suitable material for bone scaffold applications is selected in such a way that it should be biocompatible and biodegradable. Materials such as Tri Calcium Phosphate, Polyether Ether Ketone, Hydroxyapatite, Poly L Lactic Acid, Polycaprolactone and Polyamide are preferred as suitable materials for porous scaffolds. Bone scaffolds, besides providing adequate mechanical strength, should also support cell growth after implantation. Composite materials provide a favourable combination of mechanical and bioactive properties. Hence, a polymer/ceramic composite, comprising of Polyamide (USP Class VI certified) and Hydroxyapatite that was synthesized by co-precipitation technique were used in this study. Polyamide (PA) offers good mechanical strength to the scaffold, while Hydroxyapatite (HA), being a bioactive ceramic, recruits osteoblast cells, facilitating rapid bone formation. In the present study, Polyamide: Hydroxyapatite composite mix in the range of 95:5 to 80:20 in weight percentage was used.
Unit cells of porous scaffold with different pore geometry.
Porous scaffold is a three dimensional structure which mimics the extracellular matrix properties such as mechanical strength and acts as a template for cell attachment and bone formation [15]. Apart from the biochemical behavior, the pore geometry and size play a vital role in the performance of the scaffold. So, appropriate design of scaffold model plays an important role on behavior of the porous scaffold structure [16]. CAD models of porous scaffold structure with different pore shape and porosities were modelled. The following four different pore configurations were used for this study [17]:
Cubical pore model Shifted cubical pore model Spherical pore model Shifted spherical pore model
The porosity of the structure is altered by varying the spacing between the pores. The scaffold porosity is calculated as the percentage of void volume relative to the total volume within the model. Figure 2 represents the unit cell with cubical and spherical pore. The wall thickness and unit cell size is kept constant at 400 μm and 2200 μm. The pore size is kept constant as 800 μm for all the scaffold designs. When porosity level increases the strength decreases but the tissue growth increases. It is also preferred to have a minimum pore size for the strength requirement and also for the ease in attachment for the elongating cells.
The cubical pore scaffold model has internal pore in the shape of a cube of size 800 μm and is interconnected by square channels. The spherical pore unit cell scaffold model is a symmetric model with voids in the shape of spheres with diameter 800 μm and these voids are connected by cylindrical channels. The spherical pore model was designed with the idea of getting higher load bearing capacity in curved members. The unit cells were repeated using linear pattern tool to create a 20 × 20 × 4 mm specimen suitable for compression testing. The scaffold with cubical and sperical pores were also designed with its shifted pattern in which the rows of pores are shifted by an offset distance. Figure 3 shows the detailed view of shifted scaffold design. When experiencing the human bionic loads, the scaffold configuration can have higher load bearing ability due to the shifted orientation. Also, in this arrangement the interconnected pores travel in all directions with maximum pore length of 400 μm which leads to healthy growth of tissues. This design is a novel one when compared to the conventional patterns for scaffold applications.
The scaffolds with different pore shapes need to be analyzed to select the best one with respect to the minimum factor of safety. Also the influence of porosity also needs to be premeditated. The porosity range selected for the research work is 40% to 70% based on the minimum porosity requirement for mineral diffusion and the porosity of the natural bone [18]. The porosity range is varied by altering the spacing between the pores as shown in Table 1.
The CAD model of shifted porous scaffold unit cell.
Parameter to alter the porosity
Different configurations of porous structures are initially studied and compared using finite element analysis and the suitable scaffold configuration was used for the fabrication of the specimen using Selective Laser Sintering (SLS) process. SLS is an additive manufacturing technique that uses a high power laser to melt cramped particles of plastic, metal, glass, or ceramic particles into a mass having a desired three dimensional shape. Few of the conventional methods of fabricating porous scaffolds like phase separation, melt moulding, fiber bonding, compression moulding, emulsion freeze drying and solvent casting have several barriers like poor dimensional control over shaping the pore size, pore geometry, internal architecture and surface finish. SLS is one of the best method for fabricating scaffold for tissue engineering application with greater accuracy and having control on overall scaffold geometry [19–21]. DTM 2500 Plus Rapid Prototyping(RP) machine shown in Fig. 4 is used for the fabrication of the specimen. RP machine is a computer aided system, in which CAD model of a required component is to be imported in STL data format.
A stock temperature of 125 °C was used for the trial run with 100% PA material. In the next phase, different mix of HA powder (5%, 10%, 15% and 20%) with grain size similar to that of PA were loaded onto the bed. Taking into account the lean mixture of PA/HA, the same stock running temperature was used. In the first trial run, the layers build were peeled off by the roller and after several iterations the build was good at a temperature of 132 °C. The specimens were allowed to cool for 8 hours and were then taken out. The specimens are built in three orientations (horizontal, vertical and inclined) as shown in Fig. 5, so that they can be tested to evaluate the influence of build direction on its mechanical properties. The scaffold with maximum porosity (70%) was fabricated to predict the minimum value of the mechanical strength. Compression and tensile test specimen as per ASTM D638 and D695 standards respectively were fabricated with various proportions of PA/HA (Fig. 6) to test the feasibility of fabrication using composites in an SLS machine.
DTM 2500 plus SLS machine.
Build direction of the specimen.
Fabricated specimens using SLS.
In vitro cell culture studies were carried out on the specimens to predict the growth behaviour of live cells inside the scaffold. For the in vitro cell culture studies, MG 63, human osteosarcoma cell line purchased from NCCS, was used. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 200 mM L-glutamine, 10% fetal bovine serum and 1X antibiotic. The cells were maintained at 37 °C with 5% CO2. Cells were trypsinized after attaining 80% confluency.
Cell viability
MG 63 cells were tested for their viability after incubating with the developed scaffolds for 1, 3 and 5 days. In brief, the scaffolds were seeded with approximately 10,000 cells/ scaffold/ well. After 1, 3 and 5 days, 5 mg/ml of MTT solution was added to each well and incubated for 3.5 h at 37 °C. After the incubation time, the solution were discarded and the scaffolds were air dried, followed by addition of 200 μl of Dimethyl sulphoxide (DMSO) to dissolve the insoluble formazan crystals. The plates were left in shaking for 10–15 min in dark. The absorbance was then read at 570 nm in a multiwell plate reader. Cell viability test results show that the PA scaffold supported the cell growth. However, the cell growth was less than that observed on the control (without scaffold). This suggests that PA as such does not enhance bone formation, however incorporation of HA with PA does significantly.
ALP Assay
The alkaline phosphatise(ALP) activity of the osteoblasts was tested using the p-nitophenol pyrophosphate (Sigma Aldrich) as the substrate. The assay was carried out as the protocol given by Sigma Aldrich. The alkaline phosphatase activity of the cells cultured on the Polyamide scaffold was lower than compared to the control (cells cultured without scaffold). Assays such MTT assay and ALP assay were carried out.
MTT assay and ALP assay test results (Fig. 7) show that PA/HA scaffolds gave superior results compared to pure Polyamide. Hence, PA/HA composite could be a better scaffold material compared to pure Polyamide [22].
MTT assay results for MG 63 cells cultured on the scaffolds.
The CT scan data of the femur bone of 75 kg weight obtained in DICOM image format has been converted into STL format to finalize the CAD model of the femur. Finite element modeling and analysis of human bones is quite useful in biomechanical simulations [23,24]. In this research, the femur bone (Fig. 8) is subjected to loads pertaining to various types of physical activities of human body and the finite element results are computed using ANSYS software. The Young’s modulus value of cortical and cancellous bone were retrieved from the literature [25] as 17000 MPa and 800 MPa respectively. Tetrahedral elements are employed for meshing and the number of elements in the finite element model are 1,45,613 and the details of material properties used for the analysis is given in Table 2. All translational degrees of freedom except translation in vertical direction have been constrained on the distal (bottom) end of the femur surface area and the hip contact force has been applied in the normal direction on the head of the femur. Based on the stress analysis performed on the normal human femur bone [26–28] without scaffold, the von Mises stress was found higher in the shaft region of the femur bone as shown in Fig. 9.
In order to predict the feasibility of using PA/HA scaffold in human femur, the scaffold model was introduced in the location of maximum stress as shown in Fig. 10. The porous scaffold with 70% porosity was fixed by covering 25%, 50% and 75% area of the shaft bone region as shown in Fig. 11. Loads acting at femur bone via hip joint during various physical conditions such as slow walking, fast walking, running and stair climbing conditions are given in Table 3.
CAD model of human femur.
Material properties of the normal bone
von Mises stress distribution of a normal femur bone region.
CAD model of human femur with scaffold.
Scaffold with different damaged regions.
Design of porous scaffold
Static structural analysis was performed to predict the best pore configuration based on the stress concentration in the structure [33]. A cubical porous scaffold model is created and a compressive load is applied on one of its faces [34–38]. 3D tetrahedral elements with minimum edge length of 0.2 mm were used with a maximum element of 27,168. The von-Mises stress distribution for the shifted spherical pore model in X and Y direction is shown in Fig. 12.
Table 4 gives the maximum deformation and stress value of the scaffold with different designs. The stress distribution in the cubical and spherical pore models are shown in Fig. 13. During the analysis, it is noted that the stress concentration is high on the cylindrical model and is smooth in the shifted cubical model. Hence, it is considered that the cubical shifted pore model has the best load bearing capacity. Even though the maximum stress was present in the shifted models, the difference in stress was minimum.
In order to predict the effect of porosity on the performance of the scaffold [39], the shifted cube model was analysed for different porosities from 30 to 70% and the results are tabulated in Table 5. It is found that the stress and deformation varies linearly with increasing porosity. Therefore the scaffold with maximum porosity is selected for manufacturing and to be experimented to predict the minimum value of compressive and tensile properties.
Von-Mises stress distribution in shifted spherical pore model.
Finete element results of scaffold with different pore geometries
Comparison of Von-Mises stress distribution in different models.
Results of FEA of scaffold with different porosities
The biomaterial characterized for scaffold application must be tested for its load bearing capacity. The Universal Testing Machine (UTM) used for testing the specimen is ZwickRoell make with 10 KN capacity. The UTM travel rate is set as 5 mm/min and 1 mm/min for tensile and compression test respectively. In each of the build orientations, five models were fabricated and tested for predicting the compression and tensile strength. The average results of mechanical tests for the various specimens made of 100% PA are given in Table 6. From the results of mechanical test, it is clear that the scaffold in vertical build orientation had the best mechanical characteristics [40,41].
The test for estimating the physical properties was carried out on the PA/HA scaffolds with different compositions build using vertical orientation. A maximum strength of 24.3 MPa and 28.1 MPa was obtained during tensile and compression test respectively for 80:20 PA/HA. The results of other compositions are given in Table 7 and shown in Fig. 14. It is evident that the strength increases proportional to the increase in the composition of ceramic composites(HA) particles with Polyamide PA [42,43].
Results of mechanical tests on PA specimen for various build orientations
Results of mechanical tests on PA specimen for various build orientations
Standard deviation of mechanical test on PA scaffold.
Results of tensile and compression tests
Results of mechanical testing of different proportions of PA/HA.
The results of FEA for the different physical conditions of the femur bone are given in Table 8 and are shown in Fig. 15. From the material characterization, it was found that the maximum compressive strength of 70% porous (80%PA:20%HA) fabricated scaffold is 28.1 MPa. So a femur implanted with 70% porous PA:HA scaffold covering only 25% and 50% femur area could survive the physical activity of slow walking and fast walking (Table 8). As seen from the results in Table 8 and Fig. 16, when the scaffold area is increased, the femur could satisfy for the physical activity of slow walking, fast walking and running for 75% scaffold cross section. Further improvement could be achieved on the usage of PA/HA scaffold with less porosity values (60% and 50%) which will have a higher compressive strength value.
The maximum von-Mises stress of femur bone with scaffold region for various loading conditions
The maximum von-Mises stress of femur bone with scaffold region for various loading conditions
Von-Mises stress distribution of femur bone under different conditions.
Results of von-Mises stress for various loading conditions.
Severe traumas or injuries occurring due to accidents demand bone repairs. Bone repair is done by fixing artificial scaffold made of bio compatible materials like Stainless steel and titanium. The scaffold materials should possess good load bearing capacity in the injured region till the native bone is formed. The metal implants are removed most of the times which is a painful and expensive procedure. To combat the above situation, bioresorbable ceramic composites are being developed and used. PA/HA scaffolds with shifted cubical pore configuration were fabricated using Selective Laser Sintering method in vertical build direction. The mechanical test carried out on different proportions of PA:HA specimen provides a maximum strength of 24.3 MPa and 28.1 MPa during tensile and compression tests respectively for 80%PA:20%HA composition. The fabricated scaffolds were analysed in vitro for testing their toxicity and the test yields positive results for cell growth.
In order to predict the behaviour of the above scaffold in real situation, finite element analysis of the femur bone implanted with PA/HA scaffold was carried out. The PA/HA scaffold with different proportion of damaged area (25%, 50% and 75%) and 70% porosity was implanted in the area of maximum stress location. The finite element analysis was carried out to predict the stress induced during the different human activities. From the results (Fig. 17), it is found that femur with 25% and 50% scaffold portion is suitable for that slow walking and fast walking application, whereas femur with 75% scaffold portion can be suitable for the slow walking, fast walking and running condition. As the porosity of the scaffold is reduced further, the stress induced will reduce further and thus can have a higher factor of safety. The above investigation on porous structured scaffold would be very much useful in bone tissue engineering, especially in bone repair and regeneration.
Footnotes
Acknowledgements
The authors are thankful to the Department of Science and Technology (DST), Government of India, for their support and funding for the above research, and PSG TIFAC CORE in Product Design, Coimbatore, Tamil Nadu, India for their support of the fabrication process. The author S. Jothi also gratefully acknowledges the ASTUTE (Advanced Sustainable Manufacturing Technologies), which was partly funded by the European Regional Development Fund (ERDF) through the Welsh Government, Project number: 80380, and the ASTUTE 2020 (Advanced Sustainable Manufacturing Technologies) operation, which was partly funded by the European Regional Development Fund through the Welsh Government and the participating Higher Education Institutions, Project number: 80814.
Conflict of interest
None to report.