Evaluation optimum ratio of synthetic bone graft material and platelet rich fibrin mixture in a metal 3D printed implant to enhance bone regeneration

Recently, advancements in technology have enabled the successful manufacturing of patient-specific (custom) metal implants with intricate, custom shapes for clinical surgery by integrating computed tomography (CT) image reconstruction, computer-aided design (CAD), and metal 3D additive manufacturing (commonly referred to as 3D printing). However, the production of large custom implants often leads to challenges such as bone ingrowth at the metal interface, inadequate implant strength, excessive weight, and induces stress shielding [1‐6].

To address these challenges, large custom or geometrically complex metal 3D-printed implants can undergo structural lightweight design during the design stage using topological optimization methods. Structural optimization accompanied hollow spaces within the implant and synthetic or autologous bone grafts can be considered to fill in these spaces to enhances the defect reconstruction and promotes stronger bonding with surrounding bone, ensuring long-term stability after surgery. However, no study has confirmed bone growth efficacy and feasibility between synthetic bone grafts (SBGs) and metal 3D-printed implant.

SBGs are commonly used in orthopedic surgeries to fill up bone defects [7‐11]. It provides an osteoconductive effect and is relatively inexpensive. However, the powder or cubic form of the commercialized synthetic bone grafts, which has a large porous architecture, cannot fill up the bone defect/internal space of implant tightly. The loose filling space between the bone graft and the defect/internal implant surfaces could lead to delayed union or non-union [7‐11]. Due to the chemical nature of metallic salts, SBGs have been reported to dissolve faster than the bone defect recovery [7‐14]. Serious biomechanical problems, such as bone cracks or fracture can easily arise under unfavorable stress in the bone defect area due to the non-integrity structure.

Platelet rich fibrin (PRF) is the second-generation platelet rich concentration. It contains a high amount of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transformation growth factor beta (TGF-β). PRF has already been proven to improve wound healing and enhance tissue regeneration [15, 16]. The jellylike PRF can be mixed with bone graft material to become concentrated growth factor enriched bone graft matrix, also known as sticky bone [17, 18]. This adds the viscosity characteristic to the bone graft, which eases the manipulation of bone grafts during surgery and fills bone defects completely. The polymerized PRF matrix prevents early synthetic bone graft resorption [19]. Therefore, sticky bone is ideal for securely filling irregular traumatic bone defect cavities and internal space of the 3D-printed implant. The sticky bone system has already been applied with dental implants, and has proven to improve bone growth and clinical outcome [20‐22]. However, it is still unknown how the mixing ratio of SBG material and PRF can promote good bone regeneration.

When putty-like sticky bone was considered to fill into hollow space within a large metal 3D-printed implant, the implant surface needed to design with window patterns that can allow convenient placement of the filling materials inside the device during surgery. The aim of this study is to evaluate bone graft packing effect, bone ingrowth feasibility and interaction for the PRF composite mixed with different ratio of SBGs (different sticky bone) filled into a relatively small scale hallow titanium 3D-printed implant using in vivo implantation experiments on rabbits and pigs.

The body weights of both animal models were recorded, and no drastic variation was found before sacrificing. No surgical wound infection or other complications were found at the 8th week. All animals were euthanized as scheduled. The represented x rays of intact and fractured rabbit and pig bones are shown in Tables 1 and 2, respectively. As the X-ray images show, three fractures were found among the nine rabbit bones and one fracture was found in each of the three different PRF combination groups (SBG, PRF:SBG = 1:1 and PRF:SBG = 2:1). Only one fracture was found in the pure synthetic bone graft group in the pig test.

In the rabbit histomorphometrical evaluation, blue color indicating the amount of collagen-tissue and bone growth (Fig. 6). Percentage ratios of blue color to black deduction areas were 52.73%, 63.09% and 73.80% for SBG, PRF:SBG = 1:1 and PRF:SBG = 2:1 groups, respectively (Table 3). This result indicated that SBG mixed with higher PRF ratios presented relative higher percentage of collagen-tissue/bone growth. The increased percentage were found 19.65% (about 20%) and 39.95% (about 40%) for PRF with SBG in 1:1 and 2:1 ratio, respectively (Table 3).

We applied the Masson–Goldner’s trichrome stain for the pig test and found by which the cancellous bone was stained in green, the muscle-related tissue (outer-periosteum) was stained in red, and the erythrophilic leukocytes were stained in yellow (Fig. 7). Therefore, the expected results showed bone growth trend in pigs was different from that in rabbits. The increased percentages of bone growth were 32.13% for PRF with SBG in 1:1 combination and − 8.4% for the 2:1 group when compared to the pure SBG (Table 4).

The SBG is a variety of bone substitutes are used in orthopedic operations. In this study, we adapted Bicera as a representative SBG because it is one of the most commonly used bone substitute material [23]. Furthermore, it is available in powder and can be access conveniently. The chemical components of Bicera are hydroxyapatite (60%) and ß-tricalcium phosphate (40%), both compounds were widely used as SBGs [24, 25]. The porous structure of Bicera can provide better adhering sites for PRF in forming sticky bone [26]. These features can both be found in many commercialized synthetic bone grafts. Therefore, the role of Bicera in this study can be replaced by other bone graft materials that possess similar chemical components and physical characteristics. The role of SBG material can be further replaced by autologous bone grafts, which is more ideal in promoting bone healing [27]. In the dental implanting procedure, autologous bone grafts can be obtained during the bone drilling process. Dentistry cases report on sticky bone mixed with autologous bone graft material with PRF [28]. This combination may be adapted to our implanting system in future studies.

Specific bioactive additives, such as PRP, PRGF, PRF techniques have been found to promote the capability to accelerate wound healing, regulate inflammation, and improve soft and hard tissue regeneration [29‐36]. The advantages of these bioactive additives are its autologous nature, simple collection, ease of chair-side preparation, and simple clinical application without the risks associated with allogeneic or animal derived products. Compared to the PRP, the PRF exists advantages with greater simplicity of production, absence of blood manipulation, no additives, contains more healing factors and more stem cells, less trauma. The biological properties of PRF clearly show an interesting surgical versatility and all the characteristics that can support faster tissue regeneration and high-quality clinical outcomes. The PRF is able to stimulate osteogenesis in bone environment, in addition to angiogenesis [31‐34]. The pictures in Fig. 3B–D demonstrate successful PRF and sticky bone production for the rabbits and pigs. Although reports exist on achieving PRF and sticky bone production for other types of animals [37, 38], the ideal PRF and synthetic bone graft ratio has not yet been tested.

Theoretically, higher growth factor concentration should result in faster tissue growth, according to growth factor physiology. Therefore, we tested the sticky bone combination (PRF:SBG) in 2:1 and 1:1 ratio in the rabbit in vivo test. This test determined whether the PRF, synthetic SBG and metallic holow implant combination could provide a positive effect in bone healing. The result in Table 3 indicates the positive effect from the addition of PRF and a proportional relation between the collagen-tissue and growth. When compared to the SBG group alone, the collagen growth (including bone and soft tissue) was enhanced about 20% at 1:1 ratio while as high as 40% enhancement was noted from the 2:1 ratio. However, different types of collagen would be secreted by fibroblasts, osteoblasts, and other inflammatory cells in the bone healing process. With analine blue used in rabbit tests to bind collagen in the bone matrix, smooth muscle and fibrous tissue, this result is prone to misjudgment. The blue dye area within the basket implant could be easily misunderstood as the amount of new bone growth.

Based on the rabbit in vivo test, we designed a subsequent test using Yorkshire pigs with larger bone size to expand the sample size (n = 5). Moreover, we adapted another histochemical stain the Masson–Goldner’s trichrome staining to identify differentiated tissue growing inside the hollow implant [39]. Under trichrome staining, small blood vessels were marked with red color, and found around the implant site under light microscopy, as shown in Fig. 7. The neovascularization is part of wound healing and can be enhanced by VEGF, PDGF and other factors inside PRF. Accordingly the percentage of red area is proportional to the PRF ratio, coupled with a decrease in the percentage of green area. Otherwise, leukocytes (white blood cells) stained with yellow color were found surrounding the new-grown bone tissue and the residual synthetic bone graft. This co-staining phenomenon indicated synthetic bone graft degradation, which was reported to attract erythrophilic leukocytes [25]. Newly formed lamellar bone by which was stained in green within the implants.

This finding indicated that PRF growth factors could favor in enhancing fibro epithelial tissue growth rather than bone tissue in higher concentrations. Therefore, we are able to explain the discordant results between the rabbit and pig tests. Since analine blue could bind to collagen in the bone matrix, smooth muscle and fibrous tissue, the higher percentage of blue area in the 2:1 group might represent higher vessel growth in rabbit testing. After differentiating muscle tissue from bone tissue using trichrome staining, we can conclude that sticky bone with 1:1 portion of PRF and synthetic bone graft provided better bone healing effect in the pig test.

The titanium 3D printing basket implants with outer surface lattices were used as a confining boundary to support the sticky bone structure stability before bone union. Despite femur bone fractures in three rabbits and one pig, the titanium baskets in all test subjects, including those with fractures, remained intact under X ray at the 4th and 8th weeks. This proves that the titanium basket implant was stable and strong enough to withstand the test. However, larger defects reconstructed with specific 3D printing implants may require plate-and-screw fixation to enhance the structural strength to ensure bone union in future studies [3, 40]. Bicera was chosen as the representative SBG in this study. Other types of bone grafts such as autograft, allograft or other commercial products with different components might have different results. In our study, due to the small size of the implanted SBG, it was not easy to measure the accurate absorption rate. However, it can be estimated from histological sections. Since the Bicera we used is opaque, we could measure the black area to estimate the remaining SBG. In PRF with SBG 1:1 group of the pig experiment, each sample resulted in around 4.6 mm² of black area (excluded metal bracket area), however, area occupied by SBG accounts for only half of the total area (28.26 mm²). Therefore, the proportion of SBG that still exists should be about 33% (4.6 mm²/14.13 mm²). Through such calculation we can probably estimate the resorption rate was around 67%. Our study is a single cross section histomorphometrical evaluation. Serial histomorphometrical healing process evaluations were not included.

This study successfully applied Masson’s Goldner trichrome stain to indicate that equal parts of PRF and SBG mixtures (1:1) in the titanium 3D printed hollow implant can obtain the best increased bone growth of 32.13% in the pig graft experiment.