Bone formation and degradation behavior of nanocrystalline hydroxyapatite with or without collagen-type 1 in osteoporotic bone defects – an experimental study in osteoporotic goats

doi:10.1016/S0020-1383(16)47010-5

Injury

Volume 47, Supplement 2, June 2016, Pages S58-S65

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Abstract

The intention of the current work is to assess new bone formation and degradation behavior of nanocrystalline hydroxyapatite with (HA/col-1) or without collagen-type I (HA) in osteoporotic metaphyseal bone defects in goats. After ovariectomy and special low-calcium diet for three months, 3 drill hole defects in the vertebrae of L3, L4, L5, 4 drill hole defects in the right and left iliac crest and 1 drill hole defect at the distal femur were created in three Chinese mountain goats with a total of 24 defects. The defects were either filled with one of the biomaterials or left empty (empty defect control group). After 42 days, the animals were euthanized and the samples were assessed for new bone formation using high-resolution peripheral quantitative computed tomography (HR-pQCT) and histomorphometry with 2 regions of interest. Detail histology, enzymehistochemistry and immunohistochemistry as well as connexin-43 in situ hybridization and transmission electron microscopy were carried out for evaluation of degradation behavior of the materials and cellular responses of the surrounding tissue in respect to the implants. HR-pQCT showed the highest BV/TV ratio (p = 0.008) and smallest trabecular spacing (p = 0.005) for HA compared to the other groups in the region of interest at the interface with 1 mm distance to the initially created defect. The HA/col-1 yielded the highest connectivity density (Conn.D) (p = 0.034) and the highest number of trabeculae (Tb.N) (p = 0.002) compared to the HA and the control group. Histomorphometric analysis for the core region of the initially created defect revealed a statistically higher new bone formation in the HA (p = 0.001) and HA/col-1 group (p = 0.001) compared to the empty defect group including all defect sites. This result was confirmed for site specific analysis with significant higher new bone formation for the HA group for vertebral defects compared to the empty defect group (p = 0.029). For the interface region, no statistically significant differences were found between the three groups (p = 0.08). Histology revealed a good biocompatibility without inflammatory reaction for the HA- and HA/col-1 implants with a higher fragmentation of the HA-implant compared to the HA/col-1 biomaterial and formation of new bone in the region between the biomaterial fragments by osteoblasts. Fragmentation was shown by transmission electron microscopy to be caused by multinuclear osteoclast-like cells with degradation of the implant via intracellular incorporation of degraded implant material particles. In conclusion, both nanoparticulate HA with and without collagen type-1 showed better new bone formation compared to untreated drill hole defects in metaphyseal regions of this osteoporotic Chinese mountain goat model with good biocompatibility.

Introduction

Osteoporosis and particularly osteoporotic fractures have a high impact both on the quality of life of patients and on the financial aspects of Western health care systems. Biomaterials have gained interest to enhance bone healing in osteoporotic fractures and to improve treatment outcome [1].

Among many other materials hydroxyapatite and namely nanoparticulate hydroxyapatite is a potential candidate as bone substitute material in osteoporotic bone for improvement of bone healing due to its osteoconductive effects. Nanoparticulate hydroxyapatatite with needle shaped HA crystals with a size of around 20 nm has already been investigated in experimental and clinical settings for dental and orthopaedic applications [2], [3], [4], [5], [6], [7], [8], [9], [10]. In general, good new bone formation via osteoconductivity with this type of nanoparticulate hydroxyapatite was reported. In all the above mentioned studies physiological and not osteoporotic bone was investigated.

Collagens represent 25–35% of the total body proteins and can be found in cartilage, bone and in almost all types of soft tissue [11]. Among 28 known different collagen types, collagen type-I is the most abundant in the body and in bone representing more than 90% of the organic mass in bone [12]. Collagens contain sections with the amino sequence arginine, glycine, aspartic (RGD) which was discovered as a small peptide ligand with high affinity to integrins increasing the adhesiveness of surface implants for osteoblasts via binding to those transmembrane integrin receptors [13]. Facilitated cellular attachment of pre-osteoblasts on collagen via RGD-peptides with theoretical enhancement of new bone formation is therefore of interest in the use of collagens in composite biomaterials [14].

The intention of the current study is to assess new bone formation and degradation behavior of nanocrystalline hydroxyapatite with or without collagen-type I in osteoporotic bone defects in metaphyseal bone defects in osteoporotic goats. The hypothesis is that both nanoparticulate hydroxyapatite and nanoparticulate hydroxyapatite with collagen-type I enhance new bone formation compared to empty control defects and that the additional use of collagen type-I improves new bone formation compared to plain nanoparticulate hydroxyapatite.

Section snippets

Study design

There were three different treatment groups: group I: empty defect, group II: nanoparticulate hydroxyapatite, group III: nanoparticulate hydroxyapatite + collagen type I. In three osteoporotic Chinese mountain goats, a total of 24 bone defects were created that were either filled with nanoparticulate hydroxyapatite or nanoparticulate hydroxyapatite + collagen type I or were left empty after randomisation (Table 1). In each animal, 2 defects in the left iliac crest, 2 defects in the right iliac

Induction of osteoporosis and clinical observation

BMD at the left calcaneus (n = 3) dropped from 439.8 ± 50.5 mg/mm³ to 334.3 ± 40.1 mg/mm³, and at the right calcaneus from 441.3 ± 45.0 mg/mm³ to 335.3 ± 37.3 mg/mm³ 6 months after ovariectomy indicating an osteoporotic bone status with a loss of 24 ± 2% of the initial BMD.

Full-weight bearing was achieved in all three cases in the first postoperative days and all goats survived the entire observation period without any wound healing disturbance or other problems.

High-resolution peripheral quantitative computed tomography (HR-pQCT)

In general, it was observed that the

Discussion

The current study revealed a statistically significant higher new bone formation in the defect region of the HA and the HA/col-1 group including all anatomical sites compared to the empty defect group in this osteoporotic animal model in Chinese mountain goats by histomorphometry. There were no significant differences between the HA and the HA/col-1 group for this overall evaluation. HA could also show enhanced new bone formation for site specific analysis for vertebrae defects compared to the

Conclusion

In conclusion, both nanoparticulate HA with and without collagen type-1 showed better new bone formation compared to untreated drill hole defects in metaphyseal defects of the lumbar spine, the iliac crest and in the distal femur of this osteoporotic Chinese mountain goat model. Both materials showed good biocompatibility without any inflammatory reaction and degradation via osteoclast-like multinuclear cells with intracellular uptake of the material into these cells.

Conflict of interest

The authors have no conflicts of interest.

Acknowledgment

The study was sponsored by a travel grant from Germany/Hong Kong Joint Research Scheme (Ref: G_HK009/07) of the German Academic Research Service (Deutscher Akademischer Austauschdienst (DAAD)) funded by means of the Federal Ministry of Education and Reserach of Germany (Bundesministeriums für Bildung und Forschung (BMBF)). The authors thank aap Biomaterials, Dieburg, Germany, for the provision of the OSTIM^® and OSTIM^® + collagen type-I implants.

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Bone formation and degradation behavior of nanocrystalline hydroxyapatite with or without collagen-type 1 in osteoporotic bone defects – an experimental study in osteoporotic goats

Abstract

Introduction

Section snippets

Study design

Induction of osteoporosis and clinical observation

High-resolution peripheral quantitative computed tomography (HR-pQCT)

Discussion

Conclusion

Conflict of interest

Acknowledgment

Injury

J Oral Maxillofac Surg

Biomaterials

Biomaterials

J Biol Chem

Adv Drug Deliv Rev

Bone

Ultrasound Med Biol

Acta Biomater

Hydroxyapatite paste Ostim(®), without elevation of full-thickness flaps, improves alveolar healing stimulating BMP- and VEGF-mediated signal pathways: an experimental study in humans

Clin Oral Implants Res