Study on clinical application of nano-hydroxyapatite bone in bone defect repair

Abstract

Objective: To study the clinical effect of bone defect treated with nano-hydroxyapatite(Nano-HA) artificial bone. Methods: From September 2009 to June 2012, 27 cases of bone defect were analyzed retrospectively. The position of bone defect included humerus, radius, ulna, femur, tibia and calcaneus. The range of bone defect was from 0.3 × 1.0 cm to 3 × 6.5 cm. Among them, there were 22 cases with fractures and 5 cases with tumors. All patients were treated with Nano-HA artificial bone. The ability of bone defect repair was evaluated by X-ray exams performed preoperatively and postoperatively. HSS scores were adopted for final evaluation at the latest follow-up. Results: The patients were followed up from 11 to 26 months (average of 18.5 months). No general side effects occurred. X-ray photo showed an integrity interface between Nano-HA and bone. Primary healing was obtained in all cases without any complication. Conclusion: The Nano-HA artificial bone had a good biocompatibility and could be an ideal artificial bone in the reconstruction of bone defect.

Keywords::

• artificial bone
• bone defect
• hydroxyapatite
• nano-material

Introduction

The research for an ideal bone substitute material is a major focus subject in clinical practice. Hydroxyapatite, HA (molecular formula: [Ca₁₀(PO₄)₆(OH)₂]), as the main component of the inorganic substance of bone, has a good biocompatibility and conjunctive ability with bone tissues. It is one of the most common bioactive materials for bone defect repair. However, the clinically applied HA artificial bone still has the shortcomings such as poor mechanical features, high fracturability, low compressive and fractural resistance. The HA artificial bone cannot bear load and can only be used for the defect repair of non-load-bearing bones (Zhu et al. 2010b).

With the development of biomaterials and nanotechnology, people turn to nano-hydroxyapatite (nano-HA) artificial bone with better biological properties. In order to avoid these problems, investigators have developed various kinds of materials for bone grafts which had suitable biocompatibility. At present, the popular study is nanometer bone graft material, which can be used to repair bone defects more conveniently and provide sufficient mechanical properties to ensure the normal healing of the bone defect. In this regard, the People's Second Hospital of Shenzhen and Powder Metallurgy Engineering Research Center of Central South University developed cooperatively the new type nano-HA artificial bone. On the basis of animal experiment, nano-HA material was manufactured by the method of Collosol-Flocculation to improve the mechanical property and to overcome the shortcoming of higher fragility, lower intensity to resist expression and break, poor weight-bearing. At the same time, polyporous nano-HA artificial bone with gap rate above 90% and pore sizes of 100–250 μm were produced in order to perform the animal experiment of bone defect repairing. In the early stage study, the Nano-HA porous artificial bone synthesized by us was used for repairing radius bone defects in rabbit models. The results had showed that the Nano-HA artificial bone had better bone reconduction ability and biocompatibility, it can be used in the treatment of bone defect and the Nano-HA composite was characterized by sufficient mechanical properties and osteoconductive capabilities because of the effect of nanometer (Zhu et al. 2012).

In this study, Nano-HA artificial bone was prepared and the ability and the feasibility of bone's reconstruction were evaluated in repairing of bone defect. It will contribute to further improvement of Nano-HA artificial bone used for bone substitute. This research aims to evaluate the osteogenic potential of the material in the bone defect, in order to provide foundation for the further improvement and clinical utilization of the material.

Clinical data

General information

A total of 27 cases, 22 males and 5 females, were included. Their ages were all in the range of 9–67, with the average age of 31 years. Nine cases were of tibial defect, five cases were of femur defect, six cases were of humeral defect, three cases were of radial defect and four cases were of calcaneal defect. Among all the cases, 22 cases were of bone fracture and 5 cases were of benign bone tumor defect. The range of the bone defect was 0.3 × 1.0 cm –3 × 6.5 cm. The amount of the artificial bone implanted was 7.5 ± 2.8 g.

Materials

The nano-HA bone applied in this research was prepared by using calcium nitrate and ammonium dihydrogen phosphate as the raw material; the collosol–flocculation method was used to prepare the powders, to make the average crystallite size of the pure HA smaller than 100 nm. Detecting report of the material has been shown in Table I. The powders were made into evenly distributed Nano-HA artificial bone using the wood-molding method. With the inverted microscope, scanning electron microscopy and porosity measurement (Zhu et al. 2010a), the pore diameter was measured as in the range of 100–250 μm, with the porosity rate higher than 90% (Figure 1). The structure of Nano-HA artificial bone is similar to human bone.

Figure 1. (A) Nano-HA artificial bone. (B) Nano-HA artificial bone under scanning electron microscope (SEM × 200).

Table I. Detecting report of the Nano-HA material.

Column	Demands in technology	Detection results	Conclusion
Granularity	1 ˜ 100 nm	89 nm	Acceptable
Density	< 2.0 g cm^− 3	1.1 g cm^− 3	Acceptable
Pore ratio	> 90%	93%	Acceptable
Ca/P ratio	1.62 ± 0.05	1.62	Acceptable
HA Crystal ratio	> 98%	98.5%	Acceptable
Heavy metals	As < 9 Cd < 5 Hg < 5 Pb < 30 Sum < 50	8 1 4 5 18	Acceptable
PH	6.5–7.5	6.8	Acceptable
Residual quantity of epoxy ethane	≤ 10 ug/g	2 ug/g	Acceptable
Ames teratogenesis experiments	Negative	Negative	Acceptable

Surgery method

For the patients with metaphysical fracture of limb, the fracture was exposed firstly. A bone window of 2.0 × 2.5 cm was opened at 2.5–3 cm under the compression fracture. Through the bone window, the compressed articular facet was restored. The bone defect was filled with sufficient Nano-HA strip bone graft. After the compression, the cortical bone firstly cut down was attached to cover it. The fracture was fixated by the minimally invasive locking plate or the anatomic plate. For the patients with middle part of limb or calcaneal fracture, the fracture was firstly restored after the exposure of the fracture position. The bone defect was filled with Nano-HA artificial bone and internal fracture fixation was conducted (Figure 2).

Figure 2. (A) Exposing fracture of tibial plateau and bone defect. (B) Implanting Nano-HA artificial bone.

For the five cases of benign bone tumor patients, the apoxesis of tumor necrotic tissue was first performed and rinsed with iodine. After the cleaning of the wound surface by physiological saline, the sterilized Nano-HA artificial bone was cut into different shapes to fill in the defect according to the lesions of the patient. The density was made appropriate with no oppression performed. The drainage sheet or drainage tube was placed. The wound was sutured and bandaged. In order to prevent pathological fracture in the cases with large defect, the cast was used for the external fixation. After the surgery, the observation was conducted on the patients to check for adverse reactions and the wound situation. The X-ray examination, calcium and phosphate measurement and limb function recovery examination were conducted at 1, 3 and 12 months postoperatively.

Postoperative treatment

After 24–48 h of the surgery, the drainage tube was removed. After 1 week, the joint function exercises were started. Most patients were able to exercise by themselves within 1 week to realize flexion of the knee joint to 90°. If the flexion of the knee joint by autonomous exercise after 1 week was not able to reach 90°, CPM assisted exercise was performed. At the time of discharge from the hospital, the knee flexion of all the patients could realize 90°. Within 6 weeks after the surgery, the patients should avoid weight-bearing walk on the affected limb. In 6–12 weeks, partial weight could be loaded on the limb and after 12 weeks, complete weight could be loaded.

Observation was applied on healing of wounds and X-ray exams were performed preoperatively and postoperatively. HSS score (Potocnik et al. 2011) (the United States, Hospital for Special Surgery knee score) criteria assessed knee function recovery. The total scores of 100 points, the higher the score the better knee function recovery. Excellent, 85 points or more; Good, 70–85; middling, 60–69; poor, 59 points or less.

Results

The follow-up period was in the range of 11–26 months, the average was 18.5 months. Only one case of patient reported trifle exudation from the wound. It was healed by changing dressing. The other 26 cases did not report any adverse reactions. No fever was reported after the surgery and neither swelling nor allergy was reported on the wound. The wound was healed in one stage. No complications such as incision infections occurred. The X-ray examination indicated that the Nano-HA artificial bone in the bone defect region was healed directly with the host bone. The blending was good and no gap between the artificial bone and the original bone interface was found. No adverse reaction was observed after surgery. Three months after the apoxesis of the bone tumor, the bone density increased. In 13–16 months, the interface between the material and the host bone began to be fuzzy and is blended together (Figure 3).

Figure 3. (A) Preoperative: fracture of left tibial plateau. (B) 3 months postoperatively: The delimitation between new bone tissue and normal bone tissue were clear. (C) 26 months postoperatively: most of the material was degraded and new bone tissue was formatted.

Discussion

The treatment of the bone defect resulted from various reasons, such trauma, tumor and infection has always been a difficult problem in the medical domain. For long, people have tried various methods, but these methods all have certain problems and shortcomings (Zhu et al. 2008). For example, if the autologous bone is used, a second operation field should be added. The bone cut down need repairment and shaping, which prolongs the operation time and brings extra wounds for the patients. Furthermore, the autologous bone is limited and cannot be supplied in large quantity and susceptible to absorption. The process of the establishment of blood supply and the formation of new bone are slow. Low resistance to infection would lead to the failure of the operation (Chang et al. 2000). The allogenic bone implanting in the bone defect regions avoids the opening of second operation field and solves the limited bone source. However, the allogenic bone is easy to be absorbed and deformed after implantation. The antigenicity of the allogenic bone is strong, which causes the reaction, resulting in the exclusion of the implanted material influencing the treatment effects. Therefore, the development of an ideal artificial material to substitute the bone implantation to repair bone defect has become an important issue in the medical and biomaterial field. It is of great clinical significance (Jin et al. 2000).

HA is an important inorganic component of bone and tooth tissues. It has become a hot spot of biomaterial research with its good biological properties in recent years. The existing studies proved that the material had good biocompatibility. After being implanted, the material did not provoke inflammatory or foreign reactions, without teratogenic or carcinogenic effects. Since the 1950s, a profound research has been conducted on the artificially synthesized HA (Ripamonti et al. 1999). Highly pure HA single crystal was synthesized, and carbonate HA which was very close to the human bones was also synthesized. With the improvement of technology, HA products with different forms, porosity rate and degradation rate were produced. The commercially produced HA artificial bone has been authorized in the clinical application for more than 10 years with good results. Since HA artificial bone has poor mechanical properties, high factorability, it can only be used for the defect repair of non-load-bearing bone (Wang et al. 2002).

The nanotechnology is a new research field under rapid development since 1990s. Since the nano particles have unique properties, such as the surface effects, small size effects and quantum effects, the nanomaterials have unlimited potential in application (Dorner-reisel et al. 2002). The medical professionals also started research on the Nano-HA particles (or ultrafine HA powders) and found that the Nano-HA particles had stronger bioactivity than HA. Specialists (Montazeri et al. 2010) found that with smaller particle size, the twist modulus, stretching modulus and stretching strength of the bone implant are higher. The resistance to fatigue is stronger correspondingly (Zandi et al. 2010). Therefore, the synthesis of nano-grade HA can help to improve the mechanical properties of the bone implants. With the development of the biomaterials and nanotechnology, people turn to the Nano-HA artificial bone which has better biological properties. For the twist modulus, stretching modulus and stretching strength are all improved. It is generally believed that though the osteoblast can pass through pores with a diameter of 20 μm, only the pores with a diameter larger than 50 μm are helpful to the formation of bone units in the large pores. With the increase of the pore size, the bone conductivity also increases (Zhu et al. 2013). The animal in-vivo experiment indicated that with the formation of the bone tissue in the large pores, the bone tissue with Volkmann's tube or Haversian tube forms (Jose et al. 2010). The biomechanical properties of the large-pore ceramic bones were enhanced. Researchers suggest that the pore with a diameter greater than 150 μm can provide ideal place for the growth of bone tissue. Some people believe that the minimum pore diameter for the growth of bone tissue is 100 μm, while 150 μm is the ideal pore diameter (Matsuo et al. 2010). However, in-vitro experiment indicated that the increase of pore diameter and porosity rate could lower the biomechanical strength of the artificial bone. Therefore, the porous Nano-HA artificial bone with porosity rate over 90% and pore diameter between 100 and 250 μm was fabricated using the wood molding method. With the good mechanical properties of the nanomaterial, it is hoped to achieve a unification among the pore diameter, porosity rate and mechanical strength. X-ray diffraction analysis indicated that this sort of artificial bone had structure similar to natural bone. It was helpful for the growth of small and micro vessels and fiber connective tissue, inducing the crawling of the osteoblast to facilitate the osteogenesis (Kawachi et al. 2008).

During the follow-up survey of 11–26 months, the 22 cases of patients did not report fever after the surgery, and neither swelling nor allergy was reported on the wound. The wound was healed properly with no complications such as incision infections occurred. The X-ray examination indicated that the Nano-HA artificial bone in the bone defect region was healed directly with the host bone. The material can be produced domestically, with relatively lower price, which is suitable for China's circumstances. It has high availability and the surgery is easy, requiring no special equipment. Thus the application potential is broad.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study received financial support from Shenzhen Science and Technology Project (the project number is CXZZ20130321152713220 and GJHZ20130412153906739) and the Guangdong Province Medical Research Fund (the project number is B2012320) and Guangdong Province outstanding youth innovative talent training program (Seedling project, the project number is 2012LYM_0120) and Emerging Scientist Project of Shenzhen Second People's Hospital.