Analysis of Cell-Seeded 3-Dimensional Bone Constructs Manufactured In Vitro With Hydroxyapatite Granules Obtained From Red Algae

doi:10.1016/j.joms.2005.01.006

Journal of Oral and Maxillofacial Surgery

Volume 63, Issue 5, May 2005, Pages 673-681

https://doi.org/10.1016/j.joms.2005.01.006 Get rights and content

Purpose

Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering.

Materials and methods

Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician’s Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor α1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis.

Results

Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period.

Conclusion

We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Section snippets

Isolation of MCLPCs and monolayer culture

Biopsy specimens of the mandibular periosteum, obtained from 3 patients with their consent, were used to establish primary cultures of MCLPCs. Periosteal biopsy samples were dissected and placed in 10 mL of proliferation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium (1:1, Glutamax II; GIBCO, Grand Island, NY) supplemented with 10% autologous serum, 300 IU/mL penicillin G, and 300 μg/mL streptomycin sulfate. Subsequently the tissue pieces were digested in 0.25%

Results

We manufactured 72 identical constructs (24 constructs per patient, 12 per investigation day) using the same quantities, volumes, and concentrations of cell suspension (cell vitality ± SD, 88.4% ± 6.21%), fibrin glue and C GRAFT/Algipore granules, and 46 constructs without cells as negative controls. After 6 and 21 days of cultivation, 6 constructs per group were removed from the culture system to perform the following tests.

Discussion

The results of this study clearly show that 3D cell-seeded constructs of human mandibular MCLPCs/hydroxyapatite ceramic granules obtained from red algae supported the differentiation and matrix mineralization of MCLPCs when grown in a rotating system under osteogenic differentiation conditions in vitro.

The many desirable properties of hydroxyapatite [HAP: Ca10(PO₄)6(OH)2] ceramic materials used in vitro and in vivo are well documented; these include a similar composition to bone mineral,

Acknowledgments

The authors thank Laura Baumgartner for her excellent technical assistance, and Dr Doris Moser for preparing the SEM micrographs.

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: Research support was received by Bank-Austria Creditanstalt.

^⁎: Resident, Hospital of Cranio-Maxillofacial and Oral Surgery.

^†: Scientist, Hospital of Cranio-Maxillofacial and Oral Surgery.

^‡: Scientist, Hospital of Cranio-Maxillofacial and Oral Surgery.

^§: Resident, Hospital of Cranio-Maxillofacial and Oral Surgery.

^∥: Professor, Department of Otorhinolaryngology.

^¶: Resident, Hospital of Cranio-Maxillofacial and Oral Surgery.

^#: Resident, Hospital of Cranio-Maxillofacial and Oral Surgery.

^⁎⁎: Professor and Head, Hospital of Cranio-Maxillofacial and Oral Surgery.

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Scientific articlesAnalysis of Cell-Seeded 3-Dimensional Bone Constructs Manufactured In Vitro With Hydroxyapatite Granules Obtained From Red Algae

Purpose

Materials and methods

Results

Conclusion

Section snippets

Isolation of MCLPCs and monolayer culture

Results

Discussion

Acknowledgments

Biomaterials

Orthop Clin North Am

Cryobiology

Tissue Eng Orthop Surg

Biomaterials

Trends Biotechnol

J Craniomaxillofac Surg

J Oral Maxillofac Surg

Int J Oral Maxillofac Surg

Biomaterials

Biomaterials

Biomaterials

Oral Surg Oral Med Oral Pathol

Int J Oral Maxillofac Surg

Otolaryngol Clin North Am

Bone

Bone graft and bone graft substitutesA review of current technology and applications

J Appl Biomat

Hydroxyapatite granules and blocks as an extracranial augmenting material in rhesus monkeys

J Oral Maxillofac Surg

Autogenous bone grafts versus alloplastic material in maxillofacial surgery

Clin Plast Surg

Bone tissue engineering

Nat Med

Scientific articles
Analysis of Cell-Seeded 3-Dimensional Bone Constructs Manufactured In Vitro With Hydroxyapatite Granules Obtained From Red Algae