Elsevier

Injury

Volume 42, Issue 8, August 2011, Pages 814-820
Injury

Human adipose derived stem cells reduce callus volume upon BMP-2 administration in bone regeneration

https://doi.org/10.1016/j.injury.2011.03.007Get rights and content

Abstract

Introduction

The demand for new therapeutic approaches to treat bone defects and fractures is increasing in trauma surgery and orthopaedics because the number of patients with degenerative diseases is continuously growing. “Tissue Engineering” offers promising new technologies that combine the three components – cells, growth factors and matrix. Efforts are targeted at improving and accelerating recovery, especially for long bone fractures, and reducing the risk of delayed bone healing or pseudoarthrosis. Adult human adipose-derived stem cells (ASC) can differentiate into osteoblasts in an osteogenic surrounding. Bone morphogenetic protein-2 (BMP-2) accelerates and initiates this differentiation. Fibrin, a matrix that promotes wound healing, is a promising carrier for ASCs and BMP-2.

Materials and methods

In this study, a 2 mm transcortical drill hole in the femur of male rats served as a small non-critical size defect model for fracture simulation. In vivo bone healing was investigated upon administration of the growth factor BMP-2 embedded with ASCs in a locally applied fibrin matrix. Groups with the components alone were also investigated. After 2 and 4 weeks, μCT and histology were performed to determine the bone and callus volume.

Results and discussion

After only a short period of time (2 and 4 weeks), this animal model discloses comparative information about the osteogenetic potential and bone regeneration with little effort (no osteosynthesis necessary). The most significant result found in this model is that the combination of ASCs and BMP-2 in a fibrin matrix significantly reduces callus formation after 2 weeks compared to BMP-2 alone. BMP-2 alone significantly increased callus formation. ASCs embedded alone in the fibrin matrix did not lead to increased bone regeneration.

Conclusion

Transplantation of ASC modulated the callus induction by BMP-2 to a normal volume.

Introduction

Bone defects caused by fractures or osteoporosis represent an enormous therapeutic challenge in trauma surgery and orthopaedics. The problems and difficulties resulting from trauma and operation such as blood loss, injury of blood vessels and nerves etc. is associated with a number of complications and with an increased length of treatment. So far, autologous spongiosa has been used as the gold standard for bone regeneration as well as bone substitutes such as collagen or composites.38, 48 Besides the morbidities associated with the harvesting of spongiosa, these materials can cause adverse reactions, the formation of fistula or an interference of the healing process.

Giannoudis et al. described in the “Diamond Concept of Bone Fracture Healing Interactions” the mechanical environment as an essential factor for bone restoration along with cells, growth factors and scaffolds.15

Mesenchymal stem cells are able to differentiate in different lineages such as bone, cartilage or nerve. Adult human adipose-derived stem cells can be isolated from liposuction material.1, 16 Since availability of autologous bone marrow is limited, ASC can easily be obtained as a suitable and cost-saving source without any ethical problems.33 Zuk et al. noted that these cells show a stable growth and proliferation in vitro and that they have the potential to differentiate under certain conditions into lineages including bone, cartilage, fat and muscle.51 In 2004 it was already demonstrated in a clinical study that the implantation of these multipotent mesenchymal stem cells in a mixture of fibrin and autologous spongiosa could heal a defected skull bone of a 7 year old girl.23 A further advantage of the application of these autologous ASC is that they lack immunogenicity to some degree.25, 49

Fibrin, which was used in our study as a matrix, plays an important role in wound healing.2 Furthermore, fibrin shows excellent cell adhesive characteristics and it also binds and sets free growth factors.36, 42 It is an excellent scaffold material for injectable formulations.

BMPs are also well known to be osteoinductive. BMP-2 is commercially available for clinical use.40 Rosen et al. described that BMP-2 is responsible for the direct differentiation of mesenchymal stem cells in osteoblasts which accelerates and improves the bone regeneration.37 BMP-2 is a potential bone growth factor which is applied on a fleece in clinical routine.5, 8, 9 Govender et al. described 2002 a safe, effective and quick healing of open tibia fractures by applying BMP-2.17 Termaat et al. underlined the importance of local application and dosis of BMP-2 due to the fact that the dissimilation of BMP-2 takes place in a relatively short period of time.46 Besides its positive effects this signal protein and member of the transforming growth factor (TGF)-β superfamily has a significant disadvantage. On the one hand it may induce a surplus of callus and on the other hand bone may develop in muscles (heterotopic ossification).12, 24

Thus, the matrix, growth factors and cells all play an important role. However, their interaction has not been studied in detail. Therefore, we tried to investigate the role of the ASC embedded in fibrin in the interaction with BMP-2 in a load-bearing drill hole model. The purpose of our studies was to investigate a new and efficient method of bone treatment which improves and accelerates the healing process in order to reduce clinical costs and complications. The interaction of cells and mediators are known as an important factor of fracture healing.13, 14 For this reason we applied all three components of Tissue Engineering in our study.26, 34

Section snippets

Materials and methods

The animal protocol review board of the City Government of Vienna, Austria approved all experimental procedures in accordance with the Guide for the Care and Use of Laboratory Animals as defined by the National Institute of Health (MA58/05766/2007/8).

A transcortical 2 mm non-critical size bone defect was created under sterile conditions bilaterally in the middle of the femur diaphysis of 50 male Sprague–Dawley rats (Animal Research Laboratories, Himberg, Austria) (Fig. 1A). The rats weighing

Micro Computer Tomography (μCT) analysis

μCT data after 2 weeks showed no significant difference in both BV/TV and trabecular thickness in the cortical area and the medullary area in all five study groups. Increased callus formation was detected in the group F + BMP-2 (p < 0.05) after 2 and 4 weeks compared to all other groups. It could be possible that BMP-2 induced the periosteal callus formation due to heterotopic ossification.

In the medullary area the BV/TV significantly decreased at the 4 weeks time point (p < 0.05). Group F + BMP-2

Discussion

Bone defects resulting from trauma surgery and orthopaedics arise posttraumatically, after an infection, when a tumour develops or in the case of aseptic osteolysis. Transplantation of allogenous and autogenous bone are frequently employed, but a certain risk remains. Complications lead to reoperation, prolonged duration of surgery, local complications (haematoma, infection), intra- and postoperative problems, non-healing of the implant, higher costs, long rehabilitation periods and hospital

Conclusion

In our studies a 2 mm drill hole in the femur of male rats served as a small defect model that was used for fracture simulation.

After only a short period of time (2 and 4 weeks) this small animal model discloses comparative information about bone regeneration with little effort (no osteosynthesis necessary). The most significant conclusion found from this model with the help of micro-computed tomography and descriptive histology is that the combination of ASCs and BMP-2 in a fibrin matrix

Conflict of interest

None.

Acknowledgements

We thank our colleagues Karin Hahn, Christoph Castellani, Asmita Banerjee, Daniela Dopler, Tatjana Morton for their support during the completion of the project. We also want to express our gratefulness to Monika Großauer, Susanne Kerbl and Mohammad Jafamadar for their help during the preparations of the manuscript, James Ferguson for linguistic corrections as a native English speaker and Ilse Jung for statistically analysing our data.

This work was carried out under the scope of the European

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