preparation and in vitro differentiation of mesenchymal stem cell-encapsulated hydrogels for bone formation

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

Autologous bone is still the gold standard for repairing bone defects due to trauma, congenital defects, and surgical resection. However, autogenous bone grafting has significant limitations, including donor pain, risk of infection, and limited bone volume that can be isolated from the patients1,2,3,4. Numerous biomaterials have been developed as bone substitutes, combining natural or synthetic polymers with mineralized materials such as calcium phosphate or hydroxyapatite5,6. Bone formation in these engineered materials is usually achieved using the mineralized material as a priming material to allow stem cells to differentiate directly into osteoblasts through the intramembrane ossification (IMO) process7. This process lacks the angiogenic step, resulting in insufficient in vivo vascularization of the graft after implantation8,9,10, and therefore, approaches using such a process may not be optimal for treating large bone defects11.
Strategies applied to recapitulate the endochondral ossification (ECO) process, an innate mechanism in skeletogenesis during development, have been shown to overcome significant problems associated with traditional IMO-based approaches. In ECO, chondrocytes in the cartilage template release vascular endothelial growth factor (VEGF), which promotes vascular infiltration and remodeling of the cartilage template into bone12. The ECO-mediated approach to osteogenesis via cartilage remodeling and angiogenesis, which is also activated during fracture repair, uses artificially created cartilage tissue derived from MSCs as a priming material. Chondrocytes can tolerate hypoxia in bone defects, induce angiogenesis, and convert a vascular-free cartilage graft into angiogenic tissue. Numerous studies have reported that MSC-based cartilage grafts generate bone in vivo by implementing such an ECO program13,14,15,16,17,18,19,20,21.
An essential requirement for the clinical application of this ECO-mediated approach is how to prepare the desired amount of cartilage graft in a clinical setting. Preparing clinical cartilage of a size that fits the actual bone defect is not practical. Therefore, graft cartilage must form bone integrally when multiple fragments are implanted22. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification. Many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo23,24,25,26,27,28,29,30,31,32; however, the optimal support material to meet the clinical application requirements has remained undetermined. Hyaluronic acid (HA) is a biodegradable and biocompatible polysaccharide present in the extracellular matrix of cartilage33. HA interacts with MSCs via surface receptors such as CD44 to support chondrogenic differentiation25,26,28,30,31,32,34. In addition, HA scaffolds promote IMO-mediated osteogenic differentiation of human dental pulp stem cells35, and scaffolds combined with collagen promote ECO-mediated osteogenesis36,37.
Here, we present a method for preparing HA hydrogels using bone marrow-derived adult human MSCs and their use for hypertrophic chondrogenesis in vitro and subsequent endochondral ossification in vivo38. We compared the characteristics of HA with those of collagen, a material widely applied in bone tissue engineering with MSCs and a useful material for scaling up artificial grafts for endochondral ossification17. In an immunocompromised mouse model, HA and collagen constructs seeded with human MSCs were evaluated for in vivo ECO potential by subcutaneous implantation. The results show that HA hydrogels are excellent as a scaffold for MSCs to create artificial cartilage grafts that allow bone formation through ECO.
The protocol is divided into two steps. First, constructs of human MSCs seeded on hyaluronan hydrogel are prepared and differentiated into hypertrophic cartilage in vitro. Next, the differentiated constructs are implanted subcutaneously in a nude model to induce endochondral ossification in vivo (Figure 1).

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Our research intends to develop a method that improves the lack of blood flow and can be applied to large bone defects. The protocol presented here is one with the approaches we have taken to this end. The approach to bone formation using artificial cartilage the structure that mimics the natural process of skeletal development during the embryogenesis.This approach is relatively new in bone regeneration research. The advantage we inducing cartilage neogenesis, which is not an existent method currently used in clinical practice exists for vascularized bone grafts. Traditionally, bone regeneration research has used the techniques that combine stem cells, biomaterials, and growth factors alone or in combination.The method of bone formation by implanting artificial cartilage uses a techniques that makes artificial cartilage by mixing stem cells with biomaterials that serve the scaffolding material, and inducing cartilage differentiation. Healing our patients'bone defect clinically requires a large quantity of high quality artificial cartilage. Our protocol addresses the challenge of producing high quality artificial cartilage, and how to make multiple cartilage fuse with each other to form a unified bone.Our protocol helps generate larger, higher quality cartilage with fewer cells than protocols that use or do not use other scaffold materials to create artificial cartilage. Furthermore, the protocol can generate one integrated piece of bone, even with multiple pieces implanted in vivo. This is important because preparing clinical cartilage of a shape and size that fits the bone defect is impractical.

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Preparation and In Vitro Differentiation of Mesenchymal Stem Cell-Encapsulated Hydrogels for Bone Formation  

Begin by trypsinizing the human mesenchymal stem cells to create a cell suspension. Next, aliquot the cell suspension into a 1.5-milliliter tube to create 10 constructs. After centrifuging the suspension at 220g for three minutes, use a vacuum pump to remove the supernatant.Now, place the cell pellet on ice. Next, bring the thiol-modified hyaluronic acid, thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature. Under sterile conditions, use a needle equipped syringe to add one milliliter of degassed water to the bottle containing hyaluronic acid.Vortex the solution, occasionally heating it to 37 degrees Celsius until it becomes clear. Then, place the solution on ice. Under sterile conditions, add 5 milliliters of degassed water to the crosslinker bottle.Dissolve by inverting repeatedly before placing it on ice. Now, add 120 microliters of the dissolved hyaluronic acid solution to the aliquoted cell pellet and resuspend the cell pellet by pipetting the solution back and forth. Add 30 microliters of the dissolved crosslinker solution into the tube with the resuspended cell pellet.Ensure proper mixing by tapping on the tube. After mixing, briefly spin down the solution. Drop 15 microliters of the combined solution onto a paraffin-coated 24-well plate.Allow it to solidify at 37 degrees Celsius for 30 minutes. For the in vitro differentiation, add 0.5 milliliters of chondrogenic differentiation medium to the cell seated hydrogels. After three weeks, switch to the hypertrophic differentiation medium, adding 0.5 milliliters per well.Chondrogenic differentiation resulted in sulfated glycosaminoglycans in type 2 collagen positive extracellular matrix in both the hyaluronic acid or, HA, and collagen constructs. However, the distribution was more homogenous in the hyaluronic acid constructs. The collagen constructs demonstrated peripheral cells with heterogeneous morphology.The average roundness of cells in the HA constructs was higher than in the collagen constructs. Calcium deposition was detected at the outer edges of both constructs at five weeks. In the in vivo HA constructs, all implants were attached to each other in subcutaneous pockets and formed integrated bone tissue.40%of the in vivo collagen constructs were independent. Osteoid tissue with lamellar morphology was formed in the outer regions of both in vivo constructs at eight weeks post-implantation. Loss of cartilage was also observed in the HA construct.Multiple in vivo HA constructs appeared to form integrated bone tissue as indicated by the connection to the bone tissues and the presence of joint fibrous tissue. While 40%of the collagen constructs were united, the remaining constructs existed apart or were only attached without bony tissue connections. Both constructs showed positively labeled vessels in the bone marrow, and the inner cartilage was surrounded by multinucleated osteoclast cells.The mineral was deposited in the outer osteoid region of both constructs with higher mineral volume and HA constructs due to their larger size.

preparation-vitro-differentiation-mesenchymal-stem-cell-encapsulated

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JoVE Journal

Biology

94 ビュー.  Tokyo Medical and Dental University. まず、ヒト間葉系幹細胞をトリプシン処理して細胞懸濁液を作成します。次に、細胞懸濁液を1.5ミリリットルのチューブに分注し、10個のコンストラクトを作成します。懸濁液を220gで3分間遠心分離した後、真空ポンプを使用して上清を除去します。次に、セルペレットを氷の上に置きます。次に、チオール変性ヒアルロン酸、チオール反応性架橋剤、ポリエチレン...