This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (grant nos. JP19K10259 and 22K10032 to MAI).

This protocol uses 4-week-old male nude mice. House four mice in a cage under a 12 h light/dark cycle at 22&#8722;24 &#176;C and 50%&#8722;70% relative humidity. All animal experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval ID: A2019-204C, A2020-116A, and A2021-121A).
1. Preparation of buffers and reagents
<ol>
	<li>Prepare mesenchymal stem cell growth medium (MSC growth medium) by adding the supplement kit of MSC growth medium to MSC basal media and store it at 4 &#176;C.</li>
	<li>Prepare Dulbecco&#39;s Modified Eagle Medium and Ham&#39;s F12 (DMEM/F-12) medium by adding 10% fetal bovine serum (FBS), 50 &#181;g/mL gentamicin, 200 &#181;M L-alanyl-L-glutamine, and 1 ng/mL basic fibroblastic growth factor to the medium and store it at 4 &#176;C.</li>
	<li>Prepare chondrogenic medium by adding 1% ITS-G supplement, 0.12% bovine serum albumin, 50 &#181;g/mL gentamicin, 0.35 mM L-proline, 100 nM dexamethasone, and 10 ng/mL TGF-&#946;3 to DMEM just before use.</li>
	<li>Prepare hypertrophic medium by adding 10 mM &#946;-glycerophosphate, 0.12% bovine serum albumin, 10 nM dexamethasone, 200 &#181;M ascorbate-2-phosphate, 50 nM L-thyroxine, 50 &#181;g/mL gentamycin, and 50 pg/mL IL-1&#946; to DMEM just before use.</li>
	<li>Coat a 24-well culture dish with 200 &#181;L of melted paraffin pre-heated at 60 &#176;C. Allow to stand at room temperature until the paraffin is set.</li>
</ol>
2. Expansion of human MSCs
NOTE: Prior to starting the experiments, MSCs with a high potential for MSC chondrogenesis should be selected in micro mass culture as described in17.
<ol>
	<li>According to the manufacturer&#39;s instructions, culture primary human bone marrow-derived MSCs (passage 2) with MSC growth medium in a 10 cm dish at a density of 5 x 103 cells/cm2 at 37 &#176;C in 5% CO2 and 95% humidified air incubator.</li>
	<li>Change the medium every 2-3 days. Once the cells reach 80%-90% confluency, aspirate the medium from the cell culture dish using a vacuum pump, wash with 5 mL of PBS, and then aspirate the solution with a vacuum pump.</li>
	<li>Add 1 mL of 0.05% trypsin/0.02% EDTA solution to the dish. Incubate the dish for 5-10 min at 37 &#176;C.</li>
	<li>Add 1 mL of MSC growth medium to stop the enzymatic reaction. Transfer the cell suspension to a 50 mL tube.</li>
	<li>Combine the cell suspension from other dishes in the 50 mL tube and keep it on ice.</li>
	<li>Use the cell counter to count the cells by mixing 10 &#181;L of the cell suspension with 10 &#181;L of 0.4% trypan blue stain.</li>
	<li>Centrifuge the remaining cell suspension at 220 x g for 3 min at room temperature. Remove the supernatant and add cryopreservation solution at a concentration of 1.0 x 106 cells per mL.</li>
	<li>Dispense 1 mL of the cell suspension into each cryotube and store at -80 &#176;C for 12 h before transferring to liquid nitrogen. The resulting frozen stocks (passage 3) are used for this protocol.</li>
	<li>For the experiments, seed the stocked MSCs in a 10 cm dish at a density of 5 x 103 cells/cm2. Culture cells until 80%-90% confluent (passage 4) in DMEM/F-12 medium.</li>
</ol>
3. Preparation of MSC-encapsulated hydrogels
<ol>
	<li>Trypsinize the cells and prepare cell suspension as described in steps 2.3 - 2.6.</li>
	<li>To make 10 constructs (2.5 x 105 cells per construct), aliquot the cell suspension containing 2.5 x 106 cells into a 1.5 mL microtube.</li>
	<li>Centrifuge the suspension at 220 x g for 3 min, remove the supernatant with a vacuum pump, and keep it on ice.</li>
	<li>Bring the thiol-modified hyaluronic acid (HA), thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature.</li>
	<li>Under sterile conditions, add 1.0 mL of degassed water to the HA-containing bottle (see manufacturer&#39;s instructions) using a syringe with a needle.</li>
	<li>Vortex occasionally warming to 37 &#176;C until the solution becomes clear, then place on ice. It takes less than 30 min for the solids to dissolve completely.</li>
	<li>Under sterile conditions, add 0.5 mL of degassed water to the crosslinker bottle using a syringe with a needle. Dissolve by inverting several times, then place on ice.</li>
	<li>Add 120 &#181;L of the dissolved HA solution to the aliquoted cell pellet (2.5 x 106 cells). Resuspend the cell pellet by pipetting back and forth.</li>
	<li>Add 30 &#181;L of the dissolved crosslinker solution to the tube containing the HA and cells (step 3.8) and combine by tapping the tube, and then spin down briefly. Combine the HA solution and the crosslinker solution in a 4:1 ratio.</li>
	<li>Drop 15 &#181;L of the combined solution containing MSCs (2.5 x 105 cells) onto the paraffin-coated 24-well plate and allow it to solidify at 37 &#176;C for 30 min (Figure 2). Due to high viscosity, prepare at least 10% extra amount of the cell/hydrogel mixture than needed. 
	&#8203;NOTE: The characteristics of the hydrogel have been described previously39.</li>
</ol>
4. In vitro differentiation conditions
<ol>
	<li>Add 0.5 mL of chondrogenic differentiation medium to a construct of MSC-seeded HA hydrogels. Change the medium every 2-3 days.</li>
	<li>After 3 weeks, switch to hypertrophic differentiation medium (0.5 mL per well) and culture the constructs for an additional 2 weeks.</li>
</ol>
5. In vivo implantation of MSCs
<ol>
	<li>After a total of 5 weeks of in vitro culture, implant the constructs subcutaneously into the back of nude mice as described previously38,40.</li>
	<li>Weigh mice and administer a combination anesthetic prepared with 0.75 mg/kg body weight (b.w.) medetomidine, 4.0 mg/kg b.w. midazolam, and 5.0 mg/kg b.w. butorphanol by intraperitoneal injection as described38.</li>
	<li>Confirm adequate anesthesia by immobility to surgical stimuli and monitor respiratory depth by slow, regular chest movements and proper oxygen supply by the color of the pink mucosa periodically during the operation. Use veterinary ointment on the eyes to prevent dryness during anesthesia.</li>
	<li>Place the mouse on a sterile surgical drape in a prone position, sterilize the incision site with 50 ppm hypochlorous acid water (pH 5.0; HAW), and place a perforated surgical drape over the mouse. 
	&#8203;NOTE: Sterilize all surgical materials in an autoclave, ethylene oxide gas, or HAW. The surgeons should wash their fingers and wear surgical gowns and gloves.</li>
	<li>Make two 5 mm long skin incisions 2 cm apart in the shoulder and hip areas along the center line of the spine by using scissors.</li>
	<li>Insert a spatula subcutaneously through the incision to create a subcutaneous pocket.</li>
	<li>Insert two to three constructs (~15 &#181;L each, step 5.1) into each pocket with forceps. HA constructs implanted in the same pocket are prone to fusion very frequently. To prevent fusion, insert one HA construct in each pocket.</li>
	<li>Close the incisions with 4-0 braided silk sutures. Inject the mouse with an antagonist to anesthetics prepared with 0.75 mg/kg b.w. atipamezole as described38.</li>
	<li>While the mice recover from anesthesia, place the mice in sterile recovery cages containing sterile floor bedding without food and water bottles and continuously monitor body temperature, pulse, and respiration.</li>
	<li>Do not leave the mice unattended until it has regained sufficient consciousness to maintain sternal recumbency. After full recovery, return the animals to the breeding room with the other animals.</li>
	<li>Monitor the animals every couple of days during the healing period for any possible complications. Euthanize the mice by carbon dioxide inhalation with little suffering at 4- and 8-weeks post-implantation.</li>
	<li>Incise the skin at the grafted sites and remove the implanted constructs with forceps. Fix the constructs in 4% paraformaldehyde for 16 h and then subject them to analysis.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Report quantitative data as mean &#177; standard deviation (SD). Perform statistical analysis using commercial software.</li>
	<li>Use the Student&#39;s t-test or one-way ANOVA followed by Tukey&#39;s multiple comparisons test to determine significant differences between groups. p&#60;0.05 and p&#60;0.01 indicate significant differences between two groups.</li>
</ol>

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

The authors have declared that no competing interests exist.

Using appropriate scaffold materials that promote the transition from hypertrophic cartilage to bone is a promising approach to scale up MSC-based engineered hypertrophic cartilage grafts and treat bone defects of clinically significant size. Here, we show that HA is an excellent scaffold material to support the differentiation of MSC-based hypertrophic cartilage tissue in vitro and to promote endochondral bone formation in vivo38. Furthermore, in vivo, HA constructs wer...

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).

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		<tr>
			<td>0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>209-16941</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbate-2-phosphate</td>
			<td>Nacalai Tesque</td>
			<td>13571-14</td>
			<td></td>
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		<tr>
			<td>Bambanker</td>
			<td>GC Lymphotec</td>
			<td>CS-02-001</td>
			<td></td>
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		<tr>
			<td>basic fibroblastic growth factor</td>
			<td>Reprocell</td>
			<td>RCHEOT002&#160;</td>
			<td></td>
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		<tr>
			<td>bovine serum albumin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>012-23881</td>
			<td>7.5 w/v%</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>dexamethasone</td>
			<td>Merck</td>
			<td>D8893</td>
			<td></td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dormicum</td>
			<td>Astellas Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Merck</td>
			<td>D6429</td>
			<td>high glucose</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Merck</td>
			<td>D6421</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>1676045</td>
			<td>&#160;10 mg/mL</td>
		</tr>
		<tr>
			<td>Haccpper Generator</td>
			<td>TechnoMax</td>
			<td>CH-400-5QB</td>
			<td>50 ppm hypochlorous acid water</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HyStem Cell Culture Scaffold Kit</td>
			<td>Merck</td>
			<td>HYS020</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#223;</td>
			<td>PeproTech</td>
			<td>AF-200-01B</td>
			<td></td>
		</tr>
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			<td>ITS-G supplement</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>090-06741</td>
			<td>&#215;100</td>
		</tr>
		<tr>
			<td>L-Alanyl-L-Glutamine</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>016-21841</td>
			<td>200mmol/L (&#215;100)</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Nacalai Tesque</td>
			<td>29001-42</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Thyroxine</td>
			<td>Merck</td>
			<td>T1775</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM Mesenchymal Stem Cell Growth Medium 
			BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>165-13375</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS / pH7.4 100ml</td>
			<td>Medicago</td>
			<td>09-2051-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;3&#160;</td>
			<td>Proteintech</td>
			<td>HZ-1090</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetorphale</td>
			<td>Meiji Seika Kaisha</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visiocare Ointment</td>
			<td>SAVAVET/SAVA Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>048-34332</td>
			<td></td>
		</tr>
	</tbody>
</table>

integrated bone formation through in vivo endochondral ossification using mesenchymal stem cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

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.

MSC-encapsulated HA hydrogels were cultured in chondrogenic medium supplemented with TGF&#946;3, an inducer of chondrogenesis41 (step 4.1). We compared the properties of HA with those of collagen, which has been shown to be effective in creating MSC-based artificial cartilage grafts for endochondral ossification, as described previously38. Undifferentiated MSCs were not included as negative controls in this study because it has been demonstrated that undifferentiated MSCs r...

Watch this Scientific Journal Video about Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration at JoVE.com

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

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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.

781 Views.  Tokyo Medical and Dental University.  Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

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