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 were shown to facilitate the fusion of multiple constructs implanted in the same pocket to form a single integrated bone. Because the interaction of MSCs with scaffold materials significantly affects cartilage and hypertrophic chondrogenesis, selecting suitable scaffold material for ECOs is important for generating clinically effective tissue-engineered cartilage grafts. Cells embedded in HA hydrogels uniformly exhibit a rounded morphology characteristic of chondrocytes from the center to the periphery, indicating that hyaluronan provides an environment conducive to uniform cartilage formation throughout the hydrogel21,30. Unlike HA, collagen interacts with MSCs via integrin binding sequences (RGDs); however, the persistence of this interaction prevents MSCs from further differentiating in the chondrogenic pathway43, which may account for the non-typical chondrocyte cell morphology that is prominent in the periphery of collagen constructs.
If multiple grafts unite to form a single bone, this ECO-mediated approach can be extended to more clinically relevant bone defects. When multiple MSC-based and scaffold-free hypertrophic cartilage were applied to a single bone defect, it was reported that the cartilage grafts integrated with the bone defect. However, adjacent grafts did not integrate with each other22. The approach presented here may offer one possible solution to overcome this limitation. Compared to collagen constructs, multiple grafts of HA constructs had a greater tendency to fuse to form a single bone. Furthermore, bone marrow was formed between fused HA constructs, which may be related to the fact that HA supports uniform cartilage differentiation throughout the hydrogel because hypertrophic cartilage secretes VEGF and provides an environment for the hematopoietic stem-cell niche in bone marrow development 44.
The key to a successful experiment using this protocol is the quality of MSCs. Confirming that the MSCs to be used have sufficiently high chondrogenic differentiation potential in advance is recommended. Second, the size of each construct should not be too large. In our experience, the volume of a single construct is limited to 10-15 &#181;L. Larger constructs may result in insufficient oxygen and nutrient penetration into the constructs, leading to necrosis and poor differentiation in the center of the constructs. Therefore, gel thickness needs to be reduced to improve oxygen and nutrient penetration. In order to reduce gel thickness, it may be helpful to create thin gels on the porous membrane of Transwell inserts14,45. Alternatively, because HA constructs tend to fuse and form integrated bone, it may be adequate to implant many smaller constructs rather than a single large construct, as discussed below.
Limitations of this study include the need to accelerate the remodeling rate of the implanted tissues&#160;to facilitate bone formation.The implanted engineered tissue remained primarily in a hypertrophic calcified cartilage state even 8 weeks post-implantation. In bone formation via the ECO pathway, host-derived cells play a critical role in promoting ECO16,46. Providing additional channels in hypertrophic cartilage grafts has been reported to promote vascular invasion and graft mineralization in vivo20. Such channels serve as conduits for the infiltration of host cells, including osteoblasts, osteoclasts, and hematopoietic precursors, to form new tissue within the construct. Alternatively, it has been reported that multiple small fibrin-encapsulated pellets (&#181;-pellets) composed of chondrogenic differentiated MSCs can be implanted to form integrated bone47. Processing HA constructs into &#181;-pellets could increase the surface area surrounded by TRAP-positive osteoclasts, thus promoting the remodeling rate of cartilage tissue&#160;and enhancing osteogenesis (Figure 6)21,48. Furthermore, implantation of &#181;-pellets would increase the infiltration space of host cells, thereby facilitating angiogenesis and bone marrow development and generating integral bone formation with abundant vascularization. Thus, considering the tendency of HA constructs to fuse and create integrated bone, this &#181;-constructed bone regeneration strategy may accelerate the ECO process of hypertrophic cartilage grafts using HA hydrogels.
In conclusion, HA hydrogels are effective in scaling up tissue-engineered grafts with fewer cells and gels than collagen hydrogels. In addition, HA hydrogels have excellent fusion sensitivity and produce integrated bone with vascularization and marrow development between fused grafts. Therefore, modification of HA constructs to promote host cell infiltration and graft remodeling may lead to the development of implantable materials that can further promote vascularization and bone marrow development. With further optimization, this approach could be a promising alternative to current bone therapies in maxillofacial and orthopedic surgery.

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).

<table><tbody><tr><td>0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</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></tr><tr><td>Bambanker</td><td>GC Lymphotec</td><td>CS-02-001</td><td></td></tr><tr><td>basic fibroblastic growth factor</td><td>Reprocell</td><td>RCHEOT002&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</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&#039;s Modified Eagle Medium</td><td>Merck</td><td>D6429</td><td>high glucose</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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&amp;nbsp;</td><td>1676045</td><td>&amp;nbsp;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&amp;szlig;</td><td>PeproTech</td><td>AF-200-01B</td><td></td></tr><tr><td>ITS-G supplement</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</td><td>090-06741</td><td>&amp;times;100</td></tr><tr><td>L-Alanyl-L-Glutamine</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</td><td>016-21841</td><td>200mmol/L (&amp;times;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&lt;br/&gt; BulletKit</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>paraffin</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</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-&amp;beta;3&amp;nbsp;</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>&amp;beta;-glycerophosphate</td><td>FUJIFILM Wako Pure Chemical&amp;nbsp;</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.

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 require mineralized surfaces as a priming substrate to generate bone by osteogenic differentiation (i.e., intramembrane ossification)7.
The size of the hyaluronan hydrogels did not change throughout the in vitro culture period, as judged based on diameters measured under an inverted microscope, whereas the collagen hydrogels used for comparison began to shrink soon after the culture started. To reduce the size difference between the HA and collagen constructs after in vitro culture, the number of MSCs and the volume of the gel used to create the HA hydrogels were halved compared to those used for the collagen hydrogels. However, the wet weight of the HA constructs after 3 weeks of cartilage culture was 4x heavier than that of the collagen constructs.
Chondrogenic and hypertrophic differentiation in vitro. 
After chondrogenic differentiation (at week 3 of in vitro culture), sulfated glycosaminoglycans (sGAG)-positive (Safranin O/fast green staining; Figure 3A) and type II collagen-positive extracellular matrix was observed in both constructs, indicating that both HA and collagen hydrogels supported chondrogenesis. However, compared to the collagen construct, the distribution of sGAG, type II collagen (Col II), and type X collagen (Col X) were more homogeneous throughout the tissue in the HA constructs. Differences between the two constructs were also apparent in cell morphology: cells with a chondrocyte-like rounded morphology were distributed throughout the tissue in the HA constructs. In the collagen constructs, however, cells in the periphery showed a heterogeneous morphology, ranging from irregular/stretched to rounded morphology. Consistent with this, the average roundness of cells in the HA constructs was higher than in the collagen constructs, both in the periphery and central regions (34% versus. 77.6% (p&#60;0.01) and 78.3% vs. 100% (p&#60;0.05), respectively; Figure 3B). At 5 weeks of culture, calcium deposition was detected at the outer edges in both constructs (alizarin red; Figure 3C). Furthermore, expression of chondrogenic (Sox-9, aggrecan (ACAN), Col II), hypertrophic (Col X, matrix metallopeptidase 13 (MMP-13)), and osteogenic (type I collagen (Col I), bone sialoprotein (BSP), osteocalcin (OCN)) markers14 was detected by quantitative real-time RT-PCR, confirming the progression of chondrogenic and hypertrophic differentiation during in vitro culture (Figure 4).
Endochondral ossification of subcutaneously implanted constructs in vivo. 
Subcutaneous pockets were created on the back of nude mice, and two to three constructs were implanted in each pocket after hypertrophic differentiation (at week 5 of in vitro culture). At 4 or 8 weeks after implantation, all HA constructs were attached to each other in the implanted pockets (Table 1). In the collagen constructs, adhesion was observed in 60% of the pockets, while in the remaining pockets, the grafts were independent of each other. Hematoxylin and eosin (H&#38;E) staining revealed that in both constructs at 8 weeks post-implantation, osteoid tissue with lamellar morphology was formed in the outer regions of the constructs (Figure 5Aa,f). sGAG staining disappeared in the HA constructs, indicating the loss of their cartilage phenotype (Figure 5Ah). In both constructs, a bone marrow component containing hematopoietic cells and adipose tissue was formed between the inner cartilage and the outer osteoid tissues. In all HA constructs, the bone tissues of the fused constructs were connected and surrounded by joint fibrous tissue and bone marrow developed along the space between the two adherent constructs, indicating that multiple HA constructs tend to form integrated bone tissue (Figure 5Ag). The adhered collagen constructs were similarly integrated in two of the three fused cases, but in the third case, the bone tissue of the two constructs was not connected (Table 1 and&#160;Figure 5Ab). In both constructs, vessels identified by CD31-positive endothelial cells42 were observed in the bone marrow (Figures 5Ad,i), and the inner cartilage regions were surrounded by TRAP-positive multinucleated cells of the osteoclast lineage, indicating that cartilage tissue was subject to remodeling (Figures 5Ae,j).
Mineral was deposited in the outer osteoid region in both HA and collagen constructs (Figure 5B). While the total mineral density of the new bone was similar between HA and collagen constructs, the mineral volume was significantly higher in the HA constructs than that in the collagen constructs, which may reflect the fact that HA hydrogels did not shrink during in vitro culture, resulting in larger constructs than collagen hydrogels (Figure 5C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65573/65573fig01.jpg" /> 
Figure 1: Experimental design. The first step of this protocol is to encapsulate expanded MSCs in HA hydrogels to promote chondrogenic and hypertrophic differentiation in vitro. Constructs are cultured in chondrogenic differentiation conditions for 3 weeks, followed by an additional 2 weeks in hypertrophic differentiation conditions. The second step of this protocol is to subcutaneously implant the constructs into nude mice at week 5 of in vitro culture to undergo endochondral ossification in vivo for 8 weeks. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65573/65573fig02.jpg" /> 
Figure 2: Preparation of MSCs-seeded hydrogels. Drop modified HA/crosslinker solution containing MSCs onto a paraffin-coated 24-well plate and allow to solidify at 37 &#176;C for 30 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65573/65573fig03.jpg" /> 
Figure 3: Histological and immunohistochemical analysis of HA and collagen constructs after in vitro culture22. (A) Constructs at 3 weeks (3W) of in vitro culture were stained for sGAG (safranin O/fast green), type II collagen (Col II), and type X collagen (Col X). (B) The average circularity values of cells at 3 weeks of in vitro culture (n = 5). Open and grey bars indicate collagen and HA constructs, respectively. Groups without a common letter are statistically different (a versus b, p&#60;0.01; b versus c, p&#60;0.05). (C) Constructs at 5 weeks (5W) of in vitro culture were stained for calcium (alizarin red). All pictures were captured with the same magnification (Scale bar: 200 &#181;m). A low-magnification overview of the entire tissues is shown in the insets (Scale bar: 1 mm). Error bars represent mean &#177; standard deviation (SD). The one-way ANOVA followed by Tukey&#39;s multiple comparisons test was used to determine significant differences between groups. This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65573/65573fig04.jpg" /> 
Figure 4: Gene expression analysis of HA and collagen constructs at 3 and 5 weeks of in vitro culture. (A) Chondrogenic and hypertrophic markers. (B) Osteogenic markers. Values are presented as mean &#177; standard deviation (SD) (n = 3). Undifferentiated MSCs expressed high levels of Col I and MMP-13 and low levels of Sox-9 and Col X; they did not express (#) ACAN, Col II, BSP, and OCN. Error bars represent mean &#177; standard deviation (SD). This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65573/65573fig05.jpg" /> 
Figure 5: Immunohistochemical analysis and calcification of HA and collagen constructs post-implantation at 8 weeks. (A) Constructs were stained for Hematoxylin and Eosin (H&#38;E, a, b, f, and g), safranin O/Fast Green (c and h), endothelial cells (Cluster of Differentiation 31, CD31) (d and i), and Tartrate-resistant acid phosphatase (TRAP, e and j). (a and f) Overview of the entire tissues (scale bar = 500 &#181;m). H&#38;E, safranin O, CD31: scale bar = 200 &#181;m; TRAP: scale bar= 50 &#181;m. Arrows indicate CD31-positive endothelial cells. Abbreviations: c = inner cartilage tissue; o = outer osteoid tissue. (B) MicroCT (&#181;CT) imaging of collagen and HA constructs (main and inset image scale bars = 2 mm). (C,D) The total mineral density (C) and volume (D) of HA and collagen constructs (n = 4). ** indicates significant differences; p &#60;0.01. Four constructs were analyzed per group using &#181;CT. Error bars represent mean &#177; standard deviation (SD). Student&#39;s t-test was used to determine significant differences between groups. This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65573/65573fig06.jpg" /> 
Figure 6: Strategy for facilitating ECOs using HA-based artificial cartilage. Schematic diagram of ossification and bone marrow development without and with fragmentation (&#181;-pellets) of implanted HA constructs. (A) HA hydrogels have an excellent fusion tendency to form an integrated bone with vascularization and marrow development between fused grafts in vivo. The resulting implanted tissue, however, remained predominantly in a cartilage state even 8 weeks post-implantation. (B) Given the tendency of HA constructs to fuse to form integrated bone, processing HA constructs into &#181;-pellets may accelerate the remodeling rate of transplanted tissue and promote bone formation. Micronizing the constructs increases surface area, which may promote the resorption of cartilage tissue and the formation of bone tissue and may also promote angiogenesis and bone marrow development because of the increased space for host cell infiltration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydrogel</td>
			<td>Experiments</td>
			<td>Adhered</td>
			<td>United</td>
			<td>% United</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>5</td>
			<td>3</td>
			<td>2</td>
			<td>40</td>
		</tr>
		<tr>
			<td>HA</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 1: Unification rate when multiple constructs were implanted in a single pocket. Two or three constructs were subcutaneously implanted in a single pocket. Constructs were harvested 4- or 8-weeks post-implantation and histologically examined.

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

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

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.

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 ...

integrated-bone-formation-through-vivo-endochondral-ossification

Nanyang Technological University

Research

JoVE Journal

Biology

930 Views.  Tokyo Medical and Dental University. Ons onderzoek is bedoeld om een methode te ontwikkelen die het gebrek aan doorbloeding verbetert en kan worden toegepast op grote botdefecten. Het hier gepresenteerde protocol is er een van de benaderingen die we in dit verband hebben gevolgd. De benadering van botvorming met behulp van kunstmatig kraakbeen, de structuur die het natuurlijke proces van skeletontwikkeling tijdens de embryogenese nabootst.Deze benadering is relatief nieuw in het onderzoek naar botregeneratie. Het voordeel...

Video: Auteur in de schijnwerpers: Verbetering van botregeneratie met gevasculariseerde kunstmatige kraakbeenintegratie 

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Developmental Biology

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengineering

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Medicine

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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

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