Journal of Stem Cells Research Development & Therapy Category: Medical Type: Research Article
Improved Bone Formation by Differentiated Mesenchymal Stem Cells and Endothelial Progenitor Cells Seeded on High Concentrated Bioglass-Polylactic Acid Composite in Calvarial Rat Bone Defect
- Karam Eldesoqi1, Dirk Henrich2*, Abeer M El-Kady3, Bothaina M Abd El-Hady4, Karima M Sweify5, Borna Relja1, Christoph Nau1, Ingo Marzi1, Caroline Seebach2*
- 1 Department Of Trauma Surgery, Johann-Wolfgang-Goethe University Hospital, Frankfurt, Germany
- 2 Department Of Trauma Surgery, Johann-Wolfgang-Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
- 3 Department Of Biomaterial, National Research Centre, Al-Bohous St. Dokki, Cairo, Egypt
- 4 Department Of Biomaterial, National Research Centre, Al-bohous St. Dokki, Cairo, Egypt
- 5 Department Of Zoology, Womenâ€™s College For Arts, Science And Education, Ain Shams University, Cairo, Egypt
*Corresponding Author:Dirk Henrich
Department Of Trauma Surgery, Johann-Wolfgang-Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
Department Of Trauma Surgery, Johann-Wolfgang-Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
Received Date: Feb 24, 2015 Accepted Date: May 07, 2015 Published Date: May 25, 2015
Materials and methods: We investigated newly developed composite material consisting of Polylactic Acid (PLA), PLA and 20% Bioglass (PLA+BG 20%) or PLA and 40% Bioglass (PLA+BG 40%). These materials were seeded with either undifferentiated MSC / EPC or differentiated MSC / EPC and tested for cell adhesion and cell viability in vitro. Moreover, these composites were evaluated for bone formation in vivo. A Critical Size Defect (CSD) was made in each calvarium of 76 rats and composites were implanted. Animals were sacrificed after 14 weeks. Formation of new bone was evaluated by histomorphometry.
Results: Cell adhesion and cell viability in vitro is not significantly influenced by our tested composites, but differentiated MSC/EPC seeded onto PLA+BG40 improve significantly bone formation in a calvarial rat bone defect in vivo and represent a novel cell-based therapy for bone regeneration.
Bone tissue engineering tries to mimic the physiologic situation . The addition of osteogenic and angiogenic cells to a synthetic biomaterial increases their local density and rely on locally secreted growth and differentiation factors to induce bone formation. The biomaterials should present good biocompatibility for cell adhesion and cell viability [5,6] as well as controlled degradation kinetics to match the ratio of replacement by new tissue. Also, the biomaterials should provide an initial biomechanical support until cells generate the extracellular matrix [7,8].
Bioactive Glasses (BG) are a subset of inorganic bioactive materials, which are capable of reacting with physiological fluids to form tenacious bonds to bone through the formation of bone-like hydroxylapatite layers and the biological interaction of collagen with the material surface [9,10]. It has been found that reactions on BG surfaces lead to the release of critical concentrations of soluble Silicon (Si) and Calcium (Ca) ions, which induce favourable intracellular and extracellular responses leading to rapid bone formation . Although BG has traditionally been employed for its osteoconductive and osteostimulative properties, BG also exhibit proangiogenic potential in vitro and in vivo. Soluble dissolution products of BG up-regulate the production of numerous angiogenic factors by stimulated cells providing a potentially promising strategy to enhance early vascularisation and resultant bone formation [12-15].
However, BG, compared to cortical and cancellous bones, usually present low mechanical properties, especially in porous forms [16,17]. This disadvantage significantly limits the use of these materials in a very broad range of applications. Fortunately, one solution came from mimicking nature, which provides the inspiration to design materials with optimal organized structures under dynamically changing conditions. Many of these structures are composed of an intrinsically complex matrix based on organic and inorganic components which produce a natural hybrid material, usually referred to as composites. By combining two or more materials in a predesigned manner, a biomaterial can be created with properties that are not possible to be attained when considering each of the individual components separately .
Synthetic polymers (e.g., Poly-Lactide Acid (PLA)) have numerous advantages, such as excellent processing characteristics, which can ensure the off-the-shelf availability as well as being biocompatible and biodegradable at rates that can be tailored for the intended application [19,20]. Additionally, synthetic polymers possess predictable and reproducible mechanical and physical properties (e.g., tensile strength, elastic modulus, and degradation rate) and can be manufactured with great precision .
Thus, PLA/BG composite biomaterials present an ideal clinical solution to the limitations of traditional bone graft. But the optimal composition of PLA/BG composite for cell adhesion, cell viability and bone tissue engineering is still unknown.
Therefore, we prepared and investigated three different compositions of PLA/BG composite: Polymer (PLA), PLA+BG 20% and PLA+BG 40% (the glass content is 0, 20 and 40 wt% respectively) for bone tissue engineering in vitro and in vivo.
Moreover, recent investigations of our laboratory have focused on implantation of undifferentiated MSC and EPC seeded onto Tricalciumphosphate (TCP) which demonstrated enhanced bone regeneration and improved vascularization of critical size bone defects [22-24]. In these previous studies EPC demonstrated real angiogenic contribution. In this context it is unknown if differentiation of MSC/EPC can enhance bone formation. Thus, we hypothesized that the localized delivery of differentiated MSC / EPC onto PLA/BG composite enhance bone formation and promote bone healing in a critical-sized calvarial bone defect in rats.
The specific aims of this study were twofold:
- To compare the osteogenic potential of various concentration of bioglass in the composite: PLA; PLA+BG 20%, PLA+BG 40%
- To determine the osteogenic potential of undifferentiated MSC / EPC versus differentiated MSC / EPC.
MATERIALS AND METHODS
Characteristics of composite biomaterials: PLA, PLA+BG 20 and PLA+BG 40
Composite biomaterials were prepared by mixing polymer [poly(L-lactide) (PLA)] and Bioglass (BG) with 10 ml chloroform as follows: PLA, PLA/BG 20% (PLA+BG 20%) and PLA/BG 40% (PLA+BG 40%) biomaterial. The bioglass content was 0, 20 and 40 % by weight. These biomaterials will be referred to as PLA, PLA+BG 20% and PLA+BG 40%. Disc shaped specimens with a diameter of 5 mm and a thickness of 1 mm were cut and stored at room temperature under sterile conditions until use.
Cell isolation of rat Mesenchymal Stem Cells (MSC) from rat femur
After centrifugation (30 min, 1100 g) the cells in the interphase were collected and washed twice using PBS (10 min, 900g) containing 2% Fetal Bovine Serum (FBS). The cells were resuspended in 3 ml DMEM/F-12 and Supplements (gibcoÂ® by life technologies, Germany) and were counted.
Differentiation of rat Mesenchymal Stem Cells (MSC)
Cell seeding onto composite biomaterials
Scanning Electron Microscopy (SEM) of MSC and EPC onto composite biomaterials
Cell viability of MSC and EPC after seeding onto composite biomaterials
In each well from 24 well plate 10Âµl medium which contains either 2.5x105 undifferentiated MSC and 2.5x105 EPC or 2.5x105 differentiated MSC and 2.5x105 EPC, respectively were dropped over the biomaterial and incubated in CO2 incubator at 37Â°C for 1hour.
In order to stain MSC and EPC nuclei, cells were fixed with 3% paraformaldehyde for 10 minutes and after washing with PBS++(with calcium and magnesium), 1ÂµL 40,6-Diamidino-2-Phenylindole (DAPI; Sigma-Aldrich, Deisenhofen, Germany; final concentration 1Âµg=mL) were added to each well followed by further incubation for 15 minutes at 37CÂ°.
In order to detect EPC, cells were incubated for 1h with 2.9 Âµg/mL 1,1=-dioctadecyl-3,3,3=,3=-tetramethylindo-carbocyanine-labeled acetylated low density lipoprotein (DiLDL; Cell-Systems, St. Katharinen, Germany) in EBM supplemented with 20% FCS.
After three washes with PBS++, the biomaterials were subjected to fluorescence microscopy (Axio Observer; Zeiss, GÃ¶ttingen, Germany) in order to view DAPI-stained MSC as well as DiLDL/DAPI-stained EPC (Figure 3).
Animals and cell transplantation
|Group||Biomaterial||Cells||Animals ( n)|
|6||PLA||EPC + MSC||8|
|7||PLA+ BG20%||EPC + MSC||8|
|8||PLA+ BG40%||EPC + MSC||7|
|9||PLA||EPC + d.MSC||8|
|10||PLA+ BG20%||EPC + d.MSC||8|
|11||PLA+ BG40%||EPC + d.MSC||8|
According to the experimental groups (Table 1) the composite implants were immediately placed in the defects. Some defects were left unfilled to confirm that the defect was critical sized. The inczision was closed with a continuous suture (4-0 nylon, Ethicon, Somerville, NJ). Animals had free access to food and water and were monitored daily in the postoperative period for any complications or abnormal behaviour.
The animals were sacrificed with an overdose of pentobarbital (150mg/kg intraperitoneal) and weighed after 14 weeks. The skull bone was dissected free and removed. Bones were wrapped in gauze moistened with physiologic PBS-solution and stored at -80Â°C until preparation for histomorphometrical examination.
Histomorphometry of bone formation
Differentiation of rat Mesenchymal Stem Cells (MSC)
Adhesion of cells onto composite biomaterials
MSC and EPC viability
Histology of bone formation
When CSD remained empty, newly formed bone surrounded by an osteoid matrix rich in osteoblasts were only close to the borders of the surgical defect observed (Figure 4). The connective tissue in the central part of the defect was thinner than the original calvarium. It was well vascularized and rich in fibroplasts with oriented collagen fibers. Thus, in the histomorphometrical analysis (Figure 4) bone formed area [%] were evaluated in the empty defect (10.5 Â± 4.2) of the skull bone. Cell based therapy with seeding of undifferentiated MSC+EPC to our three tested composites, only PLA+BG40% demonstrated a significant increase of bone formation (39.5 Â± 12.1) compared to empty defect. PLA+MSC+EPC (24.6 Â± 10.3) and PLA+BG20%+MSC+EPC (30.0 Â± 8.1) showed bone formation with tendency to rise.
Optimal biomaterials for bone tissue engineering should be biocompatible, biodegradable, possess an ideal porosity for cell attachment and cell integration, respectively as well as useful biomechanical stability. Single component materials do not meet all these requirements, thus composite biomaterials are needed.
PLA is highly biocompatible with a better thermal procedure, compared to other biopolymers. The main limitations of PLA are poor toughness, slow degradation and hydrophobic properties, which results in low cell affinity . Pure bioglass is hard and brittle but offers a surface suitable for cell attachment. It is highly biodegradable and influences the local environment by releasing bioactive ions such as ionic calcium , which may lead to improved cellular responses at the implantation site .
Disadvantages of BG like lack of porosity occurred because it crystallizes during sintering. Recently, this has been overcome by understanding how the glass composition can be tailored to prevent crystallization . Procedure developments have now allowed the production of bioactive glass polymer hybrids (composite of PLA and BG, e.g., PLA+BG40%) for bone regeneration which share load with bone and are not brittle under cyclic loads [26,29-32]. In several studies, bioactive glasses are reported to be able to induce the up-regulation of genes in bone cells and their effect in enhancing bone formation . Due to their dissolution products bioactive glasses stimulating osteoprogenitor cells at the genetic level and bond with bone more rapidly than other bioceramics [11,34,35]. Moreover, early vascularization is a prerequisite for successful bone healing and Endothelial Progenitor Cells (EPC), seeded on appropriate biomaterials, can improve vascularization. In our former study, PLA+BG40% released the most calcium, and improved endothelial differentiation and vitality. This indicated that Ca2+ release improved EPC differentiation and enhanced early vascularization in critical size bone defects .
Interestingly in our present study, various BG content in PLA/BG composite (PLA; PLA+BG20%; PLA+BG40%) did not effect cell adhesion and cell viability in vitro when MSC and EPC were seeded on these biomaterials.
But, high concentrated PLA+BG40% demonstrate its osteogenic potential for bone formation. Moreover, pre-seeding this composite biomaterial (PLA+BG40%) with tissue-specific cells (MSC/EPC) prior to implant, especially when MSC are pre-differentiated, enhance bone formation significantly in vivo at 14 weeks compared to bioglass/PLA alone (cell free). This can be due both to the osteogenic and the vascular differentiation potential of MSC and EPC. In fact, cell-based therapy of MSC and EPC has been previously reported in literature in different studies [22,23,37-40] indicating a potential to provide vascularization for constructs used in bone regeneration. Our findings, that pre-differentiated MSC/EPC showed higher bone formation by trend, confirm these cell-based strategies.
These findings are in a line to Yu et al., . It is one method which is being examined to improve bone tissue regeneration. According to safety of a bioglass-polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect we observed in a previous study no side effects or complications .
One limitation of this study is that we observed at a very early time point for cell adhesion and viability, but according to our previous study it is possible to detect differences . Also 2 hours after cell seeding is in this experimental setting more practical.
Cell transplantation onto an optimal biomaterial is a promising alternative to the â€˜â€˜gold standardâ€™â€™ of autologous bone grafting to stimulate bone repair even in this presented severely compromised model of bone healing. As known a skull defect model without bone marrow inside is a severely compromised model because recruitment of progenitor cells is more difficult.
Therefore, our data support the hypothesis that this new created bioglass/PLA composite is a useful biomaterial, which improve bone formation at a critical-sized bone defect.
This work provides important insights into the interaction between cell-based therapy (EPC/MSC) and the currently available PLA/Bioglass composites. This information can be valuable for choosing which substitute to use clinically and, more importantly, for further development of these and new materials.
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Citation:Thorpe AA, Sammon C, Le Maitre CL (2015) â€˜Cell or Not to Cellâ€™ that is the Question: For Intervertebral Disc Regeneration? J Stem Cell Res Dev Ther 2: 004.
Copyright: © 2015 Karam Eldesoqi, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.