Method Article

Bone Conditioned Medium: Preparation and Bioassay

DOI:

10.3791/52707

⸱

July 8th, 2015

 ,  ,  ,  ,  , 
1Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, 2Laboratory of Oral Cell Biology, School of Dental Medicine, University of Bern, 3Department of Oral and Maxillofacial Surgery, School of Dental Medicine, Universitat Internacional de Catalunya, 4Robert K. Schenk Laboratory of Oral Histology, School of Dental Medicine, University of Bern, 5Department of Cranio Maxillofacial Surgery, Inselspital, University of Bern, 6Department of Implant Dentistry, School of Dentistry, Universidade Federal de Santa Catarina

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-β regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Autologous bone is widely used to bridge defects that occurred as a consequence of malformation, resective surgery, reconstructive trauma surgery, and prior to implant placement 1,2. Understanding the biological principles of how bone grafts support the process of graft consolidation is not only key to understand why autografts are considered to be the gold standard in reconstructive surgery, it is also bionic to the improved design of bone substitutes 3. Still, graft consolidation is faster with autologous bone compared to bone substitutes 4,5. Thus, it is imperative to reveal the molecular and cellular mechanisms that make autologous....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. BCM Preparation

  1. Obtain pig mandibles from the local butcher as fresh as possible. Place the mandibles onto a firm surface and release a full thickness flap paying special attention not to leave any soft tissue or periosteum attached to the bone. Work in a clean environment without the need to work under the flow hood.
  2. Once a full thickness flap is released, use a bone scraper to harvest the bone chips from the buccal side. Please note that the bone scraper has to be sharp. Handle firmly the bone scraper and with long movements collect the bone. Discard bone chips smaller that 1mm.
    1. To maintain native bone chips, place directly the b....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Bone Conditioned Medium is prepared from fresh porcine bone chips. General overview of the process to prepare BCM and to use biomaterials in combination with BCM is shown in Figure 1 and Figure 2 respectively. During the BCM preparation, it is important to obtain large bone chips with long movements as short movements or very small bone chips can affect the quality of the final BCM. Quality of BCM can be controlled by analyzing the gene expression of BCM target genes: ADM, PTX3, IL11, IL.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Bone-conditioned medium reflects the released activity of bone grafts during the early stages of bone regeneration. The protocol described here can be adapted to study the response of different types of cells involved in bone regeneration. Furthermore, the protocol can be used to prepare conditioned medium from processed bone or bone fillers. The methods are easy to perform and rely on a simple concept: the factors released from various native and processed bone. Understanding how BCM affects mesenchymal cells can help t.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose. Jordi Caballé-Serrano received a scholarship from the Foundation of Dental Research and Education, Basel, Switzerland.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Authors would like to thank Catherine Solioz for her skillful assistance.

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Pig MandiblesLocal bucher
Bone ScraperHu-FriedyPPBUSE2/36
Antibiotics & AntimicoticsAll life Technologies15240-062
Collagen Membranes (Bio-Gide)Geistlich
Fetal Calf SerumInvitrogen Corporation16030074
DMEMInvitrogen Corporation21885-025

High Pure RNA Isolation Kit		Roche11828665001
Transcriptor First Strand cDNA Synthesis KitRoche4379012001
PrimersMicrosynth
SYBR Green (for Q-RT-PCR)Roche4673484001
PBSRoche11666789001

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Buser, D. Long-term Stability of Early Implant Placement with Contour Augmentation. Journal of dental research. , (2013).
  2. Chiapasco, M., Casentini, P., Bone Zaniboni, M. augmentation procedures in implant dentistry. The International jour....

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Bone Conditioned MediumBone GraftsMesenchymal CellsGene Expression AnalysisQuantitative Real time PCRCollagen MembranesTGF beta SignalingBone RegenerationIn Vitro BioassayParacrine Effects

Related Articles