Bone is a remarkable organ playing critical functions in human physiology including protection, movement and support of other critical organs, blood production, mineral storage and homeostasis, blood pH regulation, multiple progenitor cells housing, and other [1]. The importance of bone becomes clear in the case of diseases such as osteoarthritis, osteomyelitis, and osteoporosis where bone does not function properly [2]. Along with traumatic injury, orthopedic surgery, and tumor resection, these lead to or induce bone defects or voids of which bone replacement is typically required. Autologous bone grafting is the gold standard in reconstructive surgery owing to high immunocompatibility. However, the concept is bound by several constraints relating to requirement of secondary surgery, limited amount of tissue that can be harvested, as well as increased risk of infection or recurrent pain [3]. To overcome these problems, tissue engineering approaches are emerging as convenient alternatives to promote the regenerative ability of the host body [4,5].

One of the most important stages of bone tissue engineering is the design and processing of a porous, biodegradable 3D structure called a scaffold. The scaffolds should provide structural support for cells and newly formed tissues by acting as a temporary extracellular matrix for natural processes of tissue regeneration and development. In the meantime, they should degrade at a rate comparable with new tissue growth [5]. Porosity is one of physical parameters for scaffolds necessary to be determined, since it provides adequate space for cell migration and expansion, and maintains the transport of nutrients and waste products [6,7]. In general, the interconnected pores of 100 µm diameter are considered as a minimum requirement [8,9]. Therefore, the improvement of biological activity and performance of bone-substitute materials and scaffolds is one of the major concerns in bone regeneration [10].

A variety of biomaterials have been shown to help repair or replace damaged bone tissue. Each has its strengths and weaknesses, and no one has been demonstrated to stand above the rest. Opportunities thus exist for the discovery of the best possible material to use [11]. Bioactive ceramics such as hydroxyapatite (HA), tricalcium phosphate (TCP), or the combination of them have drawn interest for hard tissue regeneration owing to their structural and chemical similarity with the inorganic component of bone [12]. These materials are osteoconductive because of exhibiting the capability to bond with bone and soft tissue, and stimulating bone growth [13]. HA demonstrates greater hydrophobic property, and possesses lower resorption rate than TCP. There have been advantageous in pairing HA with TCP, as some designed parameters such as degradability and surface characteristics of the engineered scaffold have been improved [14]. In addition, mechanical strength and fracture toughness of HA is ameliorated when a secondary phase of low melting point bioceramic was added on sintering [15].

Two promising approaches generally applied in bone tissue engineering to address the challenging requirements listed above include cell and acellular transplantations [16]. In the first strategy, mesenchymal stem cells (MSCs) are isolated from the patient, expanded ex vivo, seeded onto a scaffold and allowed to produce extracellular matrix (ECM) on the scaffold in controlled culture conditions, and finally implanted into osseous defect or void in the patient. In the second approach, however, a scaffold without cells is implanted immediately after injury/bone removal. MSCs are pluripotent cells capable of differentiation into a number of cell types. Also, they can be driven towards bone forming cells or osteoblasts which produce ECM within the scaffold ex vivo. In many preclinical trials, transplantation of scaffolds seeded with MSCs has been shown to enhance osteogenic capacity by integrating with native tissue faster than acellular scaffolds [17]. Despite indicating significant potential in bone tissue engineering, a primary obstacle in translating the first technique from the bench to the bedside is that the patient must wait for the bone graft to develop in vitro. Therefore, the ex vivo culture is generally supplemented with suitable osteoinductive factors, intending to promote rapid development of a ready-transplant cellular scaffold [18].

In this work, scaffolds with newly designed-TCP based ceramics were developed. The impacts of HA on structural and surface properties, as well as on mechanical strength of the scaffolds were determined. Moreover, the osteoconductive capacity of the fabricated scaffolds was evaluated in vivo in nude mice.

This study was carried out to fabricate a bone scaffold with high mechanical strength and excellent biocompatibility. The porous HA has attached mush attention as bone substitutes because of its ability to lead osteoconduction. However, one of the main drawbacks for real applications is its poor mechanical property. To overcome this problem, the combination of HA and a secondary phase bioceramic with lower melting point such as TCP was derived.

Among many fabrication methods, the sintering approach was adopted in this work in which an extremely high temperature (1230 °C) was used to consolidate HA particles. The method was found to be quite effective in obtaining highly porous structure. In addition, the minimal degradation of HA and the maximum level of strength of the resulting scaffolds was compromised. In fact, the sinter-ability of inorganic powders is strictly related to the starting morphology and chemical composition. The presence of volatile moieties, as water and carbonate, is an obstacle to powder densification and can provoke cracks and bubbles [20]. Therefore, prolonged heating at a low temperature of 500 °C was effective in yielding the resistant bioceramics. The composite of HA/TCP also exhibited high mechanical strength, consisting with the result of previous study [21]. At the temperature of sintering, the grains of HA and TCP were coalesced, obtaining a homogeneous composite as characterized by SEM (Fig. 3A). The interconnected pores within scaffold structures were introduced, as sucrose and the binder were removed upon heat treatment. By using 10% w/w sucrose, the porosity of about 40% was measurable (Fig. 3A and 3B). It was possible to change the porosity, just by increasing the sugar content on the preparation of dry mixtures. However, the products’ mechanical strength was decreased, accountable for increasing the porosity (results not shown), and be beyond the strength limit of hard tissue applications.

A denser microstructure with smaller pore size (15 ± 3 µm) was revealed by HA/TCP composites (Fig. 3A). In contrast, the microstructure of TCP scaffolds was more loosened with wider pore diameter of 43 ± 8 µm (Fig. 3B). The results suggested coalescing of the ceramic grains, as decreased residual porosity was taken place [20]. In compared to HA, the mechanical strength of the scaffolds consisting of HA and TCP was improved, while the property was deteriorated regarding to pure TCP (results not shown). This was due to the more brittle characteristic of the former bioceramic. By the current formulation and processing, mechanical problems usually encountered for load bearing applications was overcome. In addition, the porous and opened pore structures were assumed to be used in segmental bone defects [22]. Surface hydrophilicity was clearly affected by HA i.e., the decreasing was proportional to the amounts of HA supplemented in the combination (data not shown). The resulting hybrid scaffold demonstrated suitable physical and mechanical properties, drawing interest towards the in vivo investigation for its competency.

The ability of particular materials to serve as a scaffold on which bone cells can attach, proliferate, and be mineralized is called osteoconduction [5]. The prepared scaffolds were tested for such the property in nude mice in comparison with the controls, BD. Before seeding onto the scaffolds, human MSCs were prior induced with a growth stimulation factor, FGF2, and subsequently with a differentiation factor, BMP2 [18]. The seeded scaffolds were then ectopically implanted on the back of nude mice for which histological analysis was performed 4 weeks later. By aiming to evaluate early histological changes, the relatively short time period was limited for this work. There were no phagocytes observed in the tissue sections (Fig. 6A-C), suggesting that bacterial infection and tissue inflammation did not take place, and that the implanted materials were biocompatible [22]. For HA/TCP implants, they were filled with a well-established layer of bony tissue (Fig. 6B) with abundant osseous extracellular matrices (Fig. 6E). However, little new bone was detected for TCP implants (Fig. 6C and 6F). Indeed, the extracellular matrices of the control scaffolds were mostly filled with fibrous tissue (Fig. 6A and C). Thus, the osteoconductive activity was significantly distinct among the tested scaffolds in which the highest activity was revealed by the composite bioceramics. The appropriate micro-environmental conditions seemed to be established on the surfaces of HA/TCP scaffold in vivo [23]. Fig. (5B) demonstrated the formation of apatite-like crystals on HA/TCP surfaces after soaking in PBS for 2 weeks (Fig. 5B), whereas that of TCP samples was not visualized (Fig. 5C). This was supposed to associate with the partial dissolution of calcium phosphate crystals by which the super-saturation of calcium and phosphate ions in the microenvironment was generated. Then, the precipitation of carbonate hydroxyapatite was emerged by incorporating carbonate and magnesium ions present in the biological fluid into actions [24]. In accorded with the previous studies [25-27], transformation from particles to flank-like crystalline in situ was more apparent for HA biomaterial. This change was favorable for cell attachment, migration, and growth, and greatly affected the bioactive characteristics of HA/TCP composite. Moreover, the porosity of porous TCP structures was increased after soaking (Fig. 5C). This increment was estimated to exist in situ, influencing cell migration. As a result, by using the prepared scaffolds, newly formed bone tissues were detected at inner parts of the implants. Since pore diameters of the prepared scaffolds were lower than the requirement limit of 100 µm, it was possible to exclude them from testing trials early. Interestingly, the results suggested that limited pore size was not be a major obstacle affecting osteoconductive activity of the engineered scaffolds. In helping with the selected osteoinductive factors, FGF2 and BMP2, the porous HA/TCP composite scaffolds played important roles in facilitating cell attachment and the retention of infiltrated cells, as well as promoting them to form new bone. Consequently, it would be applied for general use in hard tissue regeneration.