Osteoinduction and Osteoconduction with Porous Beta-Tricalcium Phosphate Implanted after Fibular Resection in Humans

Osteoinductive properties of β-TCP remain unknown in humans. It is important to improve the bone grafts which have been the standard treatment for bone defect due to their biocompatibility and bone-healing properties. The purpose of this study was to radiologically clarify the bone forming property of β-TCP by evaluating the replacement of β-TCP by newly formed bone in the defect after fibular resection and to examine the histological features of a β-TCP specimen three months after grafting. Radiographs of 17 patients who underwent β-TCP grafting were evaluated. Osteoinductive and osteoconductive properties were assessed by examining bone formation from the remnant fibula, periosteum, and β-TCP alone. In one case, β-TCP was removed later because of postoperative complications and was evaluated histologically. Twenty two of 34 sites between the remnant fibula and β-TCP had achieved good bone regeneration. Five of 14 sites between the periosteum and β-TCP had achieved good bone regeneration. We found immature but evident bone formation in three cases with no osseous and periosteal sites. Histological analysis revealed bone formation on the outer macropore surface of β-TCP. Some blood vessels formed in the macropores expressed CD31 and CD34, while a few lymphatic vessels expressed CD34 and podoplanin. Thus, the osteoinductive ability of β-TCP alone was demonstrated in humans radiographically for the first time. The histological morphology of β-TCP was demonstrated at an early stage after grafting in humans.


Introduction
Recently, bioactive ceramics have gained popularity for filling bone defect secondary to trauma or tumor resection [1] [2] [3] [4] [5]. Autogenous bone grafts have been the standard treatment for bone loss due to their biocompatibility and bone-healing properties [6]. However, the amount of bone that can be harvested from a patient's bone is a limitation. Therefore, bioactive ceramic substitutes are a suitable option for filling large bone defects. A variety of synthetic ceramic substitutes have been developed to fill bone defects [7]. Hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), beta-tricalcium phosphate (β-TCP) (Ca 3 (PO 4 ) 2 ), and their derivatives and combinations are the most commonly used ceramic materials in bone surgeries [8].
Previously, we reported that β-TCP is a suitable bone-filling agent in clinical applications [3] [4] [5] [9] [10] [11]. Beta-TCP has been shown to have good biocompatibility and osteoconductivity in both animal experiments and clinical settings [12] [13]. Numerous basic studies have demonstrated that β-TCP enables good osteoconduction in various animals including human bone [3] [9] [13] [14]. However, calcium phosphate ceramics generally lack the osteoinductive properties needed for bone healing in large defect [15]. It has been shown that certain porous calcium phosphate bioceramics and coatings on metal implants induce ectopic bone formation when implanted in the muscles of large animals without adding osteogenic cells or bone growth factors prior to implantation [16] [17] [18] [19] [20], while the osteoinductive properties of β-TCP remain unknown in humans.
Meanwhile, fibular bone defects are sometimes seen in surgeries for tumors of bones and soft tissues because of fibular bone grafts or resections of the primary fibular tumor. Before 2004, fibular defects were not reconstructed in our institutions. On the other hand, the residual functional deficit or ankle instability had been reported following fibulectomy [21] [22]. Since 2005, highly purified β-TCP blocks have been used to fill bone defects after resection of the fibula. The purpose of this study was to radiologically clarify the osteoinductive and osteoconductive properties of β-TCP in humans by evaluating the newly formed bone in the donor sites following the use of β-TCP.
Additionally, reports of histological examination of β-TCP in human bones at early stage are rare. The secondary purpose of this study was to examine the histological features of a β-TCP specimen three months after grafting.

Patients, Materials and Methods
Between The primary objective was to radiographically evaluate the osteoconductive and osteoinductive properties of β-TCP in humans. In all patients, a plain anteroposterior radiograph of the lower leg was obtained. The immediate postoperative and the last follow-up X-rays were assessed to evaluated the bone formation of β-TCP. Three radiographic points were established in order to evaluate the osteoconductive and osteoinductive properties of the grafted β-TCP ( Figure 1).
The first point was defined by bone formation between the remnant fibula and β-TCP regardless of periosteal preservation, which was called osteogenesis from the remnant fibula. The second point, called osteogenesis from the periosteum, was defined as bone formation between the periosteum and β-TCP in cases with periosteal preservation, such as non-vascularized free fibular grafts. In this way, we evaluated the osteoconductive properties of β-TCP scaffolds to investigate directing bone formation at local osseous sites. The third point was defined by bone formation using β-TCP scaffolds alone in cases with fibular resection along Bone formation between the remnant fibula and β-TCP, which was called osteogenesis from the remnant fibula; (C) Bone formation between the periosteum (dotted line) and β-TCP in cases with periosteal preservation such as non-vascularized free fibular grafts, which was called osteogenesis from the periosteum; (D) Bone formation using β-TCP scaffolds alone in cases with vascularized fibular grafts and no periosteum at the donor site, which was called osteogenesis induced by β-TCP alone. Journal of Biomaterials and Nanobiotechnology with the periosteum such as vascularized fibular grafts, which was called osteogenesis induced by β-TCP alone. In this way, we evaluated its osteoinductive properties. Based on the radiographic findings at the last follow-up, the newly formed bone could be divided into three categories: poor (little or no bone formation at the donor site), fair (some discontinuous or immature bone formation), and good (regeneration of cortical bone with a medullary cavity) ( Table   1).
The secondary objective was to histologically evaluate the early osteoconduc-

Clinical Data of the Patients
The demographic data is presented in

Radiographic Assessment of β-TCP
We assessed the three radiographic points mentioned previously ( Table 3). The degree of osteogenesis from the remnant fibula was assessed at 34 sites between the remnant fibula and β-TCP (sum of proximal and distal remnants of the fibula). Journal of Biomaterials and Nanobiotechnology The results noted were as follows: 22 (64.7%), good; 0, fair; and 12 (35.3%), poor.
At 14 sites between the periosteum and β-TCP, the degree of osteogenesis from the periosteum was assessed, which had the following results: 5 (35.7%), good; 5 (35.7%), fair; and 4 (28.6%), poor. Good results in both these radiographic points were noted in case 17 (Figure 2), which revealed complete regeneration of the fibula radiologically. Figure 3 describes the case with poor osteogenesis from the remnant fibula (case 9). This case had absorption of the proximal and distal sides of the β-TCP blocks. In three patients who underwent resection of the fibula with periosteum, the degree of osteogenesis induced by β-TCP alone was assessed and the results were as follows: 0, good; 2 (66.7%), fair; and 1 (33.3%), poor. For example, newly formed bone with a medullary cavity was observed from the remnant proximal and distal fibula in case 1 (Figure 4). A little bone formation was    observed within the central part of the bone defect. Bone marrow aspirates were infiltrated with β-TCP blocks before implantation in this case. Poor osteogenesis induced by β-TCP alone was noted. In case 3, a regenerated medullary cavity Journal of Biomaterials and Nanobiotechnology was observed. However, bone regeneration was immature and discontinuous in the central part of the bone defect ( Figure 5). Bone marrow aspirates were also infiltrated with β-TCP blocks before implantation in this case. Fair osteogenesis induced by β-TCP alone was noted. In case 2, almost all of the grafted β-TCP was absorbed and immature regeneration of the fibular bone was observed ( Figure 6). Fair osteogenesis induced by β-TCP alone was noted. However, bone regeneration was evident in the central part of the bone defect although bone marrow aspirates were not infiltrated with β-TCP blocks before implantation in this case.

Histological Evaluation
The specimen was evaluated on an axial section (Figure 7).    TRAP and CD68. The TRAP and CD68 positive cells were mostly located in the middle region in a serial section (arrows). CD68 positive cells were scarce and TRAP positive cells were absent in the central region. Some vessels were found in every region of the specimen, although these vessels were mainly concentrated in the middle region. Most vessels in the macropores expressed CD31 and CD34 in a serial section (asterisks), while few capillaries expressed CD34 and podoplanin (arrowheads). TCP, β-tricalcium phosphate; NB, newly formed bone.
in the macropores of β-TCP. These vessels were found in every region of the specimen, although they were mainly concentrated in the middle region. Most of these vessels expressed CD31 and CD34 on a serial section, while fewer vessels expressed CD34 and podoplanin.

Discussion
The primary aim of this study was to clarify the osteoconductive and osteoinductive properties of β-TCP in human radiologically. We found satisfactory bone formation between the remnant fibula and β-TCP, and between the periosteum and β-TCP. The findings corroborated with those of previous studies Journal of Biomaterials and Nanobiotechnology that documented the regeneration of the fibula with β-TCP [25]. We evaluated the osteoconductive properties of β-TCP using two radiographic points. The osteogenesis from the remnant fibula was good in 22 (64.7%) patients but poor in 12 (35.3%). In most cases with good results, almost normal cortical bone and intramedullary space was observed in the places of β-TCP blocks. These findings suggest the process of bony ingrowth from the margins of remnant fibula over the β-TCP block that can be resorbed. The probable reason for the poor results may be the short follow-up periods, which were due to dropping out of the patients or death due to underlying diseases. There were 5 (35.7%), 5 (35.7%), and 4 (28.6%) results of good, fair, and poor osteogenesis from the periosteum. The periosteum is known to be pluripotent. It consists of osteoblastic and chondrogenic cells and it may be utilized to engineer new bone formation in vivo [26].
Autogenic periosteum could increase the bioactivity of ceramics in heterosites and improve bone formation in porous calcium phosphate ceramics [27]. good osteoconductive properties of β-TCP radiographically. However, new bone was not observed at the donor site with β-TCP in the case of patients who underwent vascularized fibular grafting [25]. In the past, osteoinduction of β-TCP without concomitant use of bone marrow cells or bone-inductive cytokines has not been reported in humans. On the other hand, porous β-TCP has been known to demonstrate osteoinductive ability in certain animals [15]- [20]. We previously investigated the process of osteoinduction in porous β-TCP in canine dorsal muscles, which suggested that the micropores on the macropore surfaces are critical for this process [10] [11]. The result suggests that the microstructure of porous β-TCP is also an important factor in the process of osteoinduction in humans. The current study is noteworthy because the osteoinductive ability of β-TCP alone was demonstrated in humans radiographically for the first time.
The secondary aim was histological examination of a β-TCP specimen in human bone. The findings demonstrated the promotion of bone formation in the axial section of the specimen. Abundant new bone formation on β-TCP was observed in the peripheral region. In this case, the periosteum was preserved at the donor site. This result suggested that the osteoblasts of the periosteum infiltrated the macropores of β-TCP and that the bone formation extended from the peri-Journal of Biomaterials and Nanobiotechnology phery to the center of β-TCP. In the middle regions, numerous multinucleated-giant cells that were positive for TRAP and CD68 were directly attached to β-TCP, thus, suggesting that β-TCP is resorbed by osteoclasts and leads to osteoblastic new bone formation over the surface of β-TCP. In immunohistochemical analysis, CD31 and CD34 positive vessels were generally found in the macropores. CD31 and CD34 are vascular endothelial marker which express in the blood vessels [28]. This finding suggested the importance of blood vessels for bone regeneration. A few vessels expressed CD34 and podoplanin. CD34 is an endothelial marker, while podoplanin is a selective marker of lymphatic endothelium [29]. This finding suggested that lymphatic vessels invaded the macropores of β-TCP along with the blood vessels. Edwards et al. reported that lymphatic vessels are absent in normal bone but are observed in pathological conditions such as lymphangioma, some primary bone tumors, and Gorham-Stout disease [30]. However, lymphatic vessels in the regenerated bone from β-TCP have not been previously reported. The current study is noteworthy because it is the first to report lymphatic vessels in β-TCP histologically, although the role was not clear for bone regeneration.
There are several limitations to this study. The number of the patients was small. This study included patients with only short-term follow-up. There were no control patients who underwent fibular resection without grafting of β-TCP at the donor site. Future studies should include more cases with a longer follow-up to corroborate these findings.
In this study, we demonstrated radiographic regeneration of bone in place of β-TCP and histological morphology of β-TCP in the early stages following

Conclusion
The osteoinductive and osteoconductive properties of β-TCP were radiologically demonstrated by evaluating the replacement of β-TCP by newly formed bone in fibular defects in humans. Beta-TCP has good osteoconductive and weak osteoinductive properties in humans. Histological examination suggested that the osteoblasts of the periosteum infiltrated the macropores of β-TCP and the bone formation extended from the periphery to the center of β-TCP. Beta-TCP is resorbed by osteoclasts and leads to osteoblastic new bone formation over the surface of β-TCP.