This project was funded by the German Research Association (grant HE 4578/3-1) and partially by the FP7 EU-Project &#x201C;GAMBA&#x201D; NMP3-SL-2010-245993.

A. Preparation of a Donor Rabbit for the Isolation of Mesenchymal Stem Cells (Surgery Room)
<ol>
	<li>Cells are isolated from male New Zealand White (NZW) rabbits at age of 4 months and approximately 3 kg body weight.</li>
	<li>Induce anesthesia by propofol (10 mg/kg body weight i.v.) and sacrifice with sodium pentobarbital (100 mg/kg body weight i.v.).</li>
	<li>Shave fur from hind limbs, back and belly with an electric clipper and vacuum the fur.</li>
	<li>Disinfect the shaved area thoroughly with 70% ethanol.</li>
	<li>Use blunt forceps, sharp scissors (or scalpel) and bone cutters for tissue and ligaments.</li>
	<li>Make an incision along the cranial surface of the leg and calf.</li>
	<li>Reflect skin and subcutaneous tissue by either sharp or blunt dissection.</li>
	<li>Separate muscles and ligaments from tibia and femur. Keep cuts as close to the bone as possible to make a clean dissection. Do not separate femur from tibia at this time.</li>
	<li>Cut through the hip joint to separate the head of the femur from the acetabulum.</li>
	<li>Elevate the tibial-femoral complex.</li>
	<li>Use the scalpel blade to scrape off any remaining soft tissue from the bones or rub the bones with sterile cloth tissues. At this point, femur and tibia are still connected.</li>
	<li>Remove patella by cutting, then cut knee joint ligaments to separate bones finally.</li>
	<li>Spray separated bones with 70% ethanol, let air dry and place each bone into a 50 ml centrifuge tube with cell culture medium (DMEM + 1% penicillin/streptomycin (Pen/Strep)) to keep them moist.</li>
	<li>Now switch under a sterile cell culture laminar flow hood.</li>
</ol>
B. Flushing of Rabbit MSC from Bones and Expansion (Cell Culture Hood)
<ol>
	<li>Collect bones from tubes and place them into 150 mm dishes using sterile forceps.</li>
	<li>Remove both bone ends with a sterile saw and move pieces to new 150 mm dishes.</li>
	<li>Fill a 10 ml syringe with medium (DMEM), attach an 18 gauge needle and insert into the opening of the bone marrow.</li>
	<li>Then, rinse marrow cavity with medium to flush the bone marrow into the dish. Afterwards, rinse from the other end, if possible. If necessary, saw off more from the ends. If bone should break, just rinse the inside of the bone.</li>
	<li>Aspirate cell medium suspension into the syringe and rinse the bone marrow repeatedly until suspension is free floating through the bone marrow cavity and no further bone marrow-clots appear.</li>
	<li>Once bone marrow has been collected from all bones, disrupt the marrow clumps by passing through an 18 gauge needle: fill syringe with needle attached and force out into medium.</li>
	<li>Afterwards, filter the suspension through a cell filter into a 50 ml tube. In order to prevent cell loss, wash culture dish 2x with 10 ml medium and filter as well.</li>
	<li>Centrifuge suspension at 500 x g for 5 min at RT.</li>
	<li>Remove supernatant and resuspend cell pellet in 10 ml medium (DMEM + 1% Pen/Strep).</li>
	<li>Separate blood cells from Peripheral Blood Mononuclear Cells (PBMC) and mesenchymal stem cells (MSC) using a Biocoll Separating Solution.</li>
	<li>Fill 5 ml of Biocoll Separating Solution into a 15 ml tube and carefully add 5 ml of cell-suspension on top and centrifuge at 800 x g for 20 min at RT (without brake).</li>
	<li>Possible results see Figure 1: Being denser than Biocoll, red blood cells sediment to the bottom while PBMC and mesenchymal stem cells remain at the interface.</li>
	<li>Carefully remove interface into a 15 ml tube and wash with 5 ml PBS.</li>
	<li>Centrifuge as described in step 22, resuspend in 5 ml PBS and repeat 2-3x.</li>
	<li>Then, centrifuge again at 350 x g for 10 min at RT (with brake).</li>
	<li>Resuspend in 10 ml medium and count cells in a hemocytometer.</li>
	<li>Plate cells at an initial seeding density of approximately 5 x 106 in 150 mm dishes.</li>
	<li>After 2-3 days, remove non-adherent cells. You might have to rinse with PBS first in order to remove cell debris. Add fresh complete medium (DMEM + 10% Fetal Calf Serum (FCS) + 1% Pen/Strep) afterwards.</li>
	<li>Feed cells every 3-4 days (Figure 2).</li>
	<li>After 5-10 days, passage the cells for the first time.</li>
</ol>
C. Preparation of Fibrin Clots in vitro
<ol>
	<li>At the day of implantation, release adherent cells from flasks/dishes by a 3 min-exposure to 0.25% trypsin-EDTA. Stop trypsinization by adding complete medium.</li>
	<li>Distribute cells in a 50 ml falcon tube and wash them twice with PBS.</li>
	<li>Determine cell viability and numbers by trypan blue staining.</li>
	<li>Add 50,000 cells/microcentrifuge tube and collect pellets by centrifugation at 500 x g for 5 min at RT. Prepare a mastermix for at least one more clot as needed.</li>
	<li>Resuspend the cell pellet in 17 &mu;l PBS and mix 25 &mu;l of the fibrinogen component of TISSUCOL-Kit with this 17 &mu;l MSC suspension.</li>
	<li>Take a sterile plate with pre-drilled holes (3x3.6 mm) in accordance to the drill holes in vivo (Figure 3).</li>
	<li>First, inoculate 4 &mu;l thrombin solution (500 IU/ml) into one hole, followed by immediate addition of 42 &mu;l fibrinogen-cell-suspension and again 4 &mu;l thrombin solution on top. Do not mix the suspension to avoid clotting in the pipette tip. First, the 50 &mu;l volume of the pipetted fibrinogen-cell-suspension will protrude the rim of the pre-drilled holes without melting due to the surface tension. However, after complete clotting (after 60 min) the clot is contracting and fits into the pre-drilled hole.</li>
	<li>Remove the clot carefully using a blunt forceps and place into a microcentrifuge tube with PBS. Prepare 2 clots/animal.</li>
	<li>Take the clots to the surgery room.</li>
</ol>
D. Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
<ol>
	<li>Induce anesthesia to rabbit (NZW, male, 3.5-4.0 kg body weight, 5-6 months old) by i.v. injection of propofol (10 mg/kg body weight).</li>
	<li>Shave the knee to be operated on with an electric clipper and vacuum the fur. All the procedures named before are performed in a surgery preparation room to avoid contamination of the sterile environment of the operating room.</li>
	<li>After intubation, maintain anesthesia with 1.5 mg/kg/min propofol and 0.05 mg/kg/min fentanyl intravenously. Monitor anesthesia by using capnography, pulse oximetry and pulse rate.</li>
	<li>Disinfect the shaved knee thoroughly and cover the rest of rabbit with a sterile dressing.</li>
	<li>Palpate the patella and perform a skin incision medial to the patella.</li>
	<li>Open the knee joint by a medial parapatellar arthrotomy under sterile conditions. Try to avoid cutting any small superficial blood vessels.</li>
	<li>Displace the patella laterally (Figure 4).</li>
	<li>After inspection of the knee joint for any concomitant cartilage lesions or joint anomalies, create two osteochondral defects (3 mm deep, figure eight-shaped) in the trochlear groove with a sterile air operating power drill (3.6 mm in diameter) with a stop-device (Figure 5) .</li>
	<li>Clean the defects and rinse them with sterile saline.</li>
	<li>Prior to implantation, fill 20 &mu;l of fibrin glue into the defects and distribute them evenly onto the bottom of the defect.</li>
	<li>Then implant the clots press-fitted into the figure eight-shaped defect.</li>
	<li>After clotting, relocate the patella within the trochlear groove and bend and stretch the knee a few times.</li>
	<li>Displace the patella laterally once again and check if fibrinogen-cell-clots are still in place.</li>
	<li>Replace the patella again and finish the operation with wound closure in layers with single button sutures (4-0 Vicryl) and a continuous cutaneous suture (4-0 Monocryl) (both with absorbable suture material).</li>
	<li>Finally, seal the wound with a spray dressing permeable to water vapor.</li>
	<li>For post-operative care, the wound is checked daily for 7 days. The rabbits receive for post-operative analgesia Carprofen 4 mg/kg s.c. every 24 hr (for 4 days) and Buprenorphin 0.03 mg/kg s.c. every 12 hr (for 2.5 days). A stabilization of the knee (e.g. dressing) is not necessary. </li>
</ol>

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+articular+cartilage+and+subchondral+bone+after+bone+grafting+and+matrix-associated+autologous+chondrocyte+implantation+for+osteochondritis+dissecans+of+the+knee.">Remodeling of articular cartilage and subchondral bone after bone grafting and matrix-associated autologous chondrocyte implantation for osteochondritis dissecans of the knee.</a> The American Journal of Sports Medicine. 39, 764-773 (2011).</li><li>Aurich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+transplantation+by+the+sandwich+technique.+A+salvage+procedure+for+osteochondritis+dissecans+of+the+knee.">Autologous chondrocyte transplantation by the sandwich technique. 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The authors have nothing to disclose.

In recent years, the possibility of treating complex articular osteochondral defects - such as those resulting from osteochondritis dissecans, osteonecrosis and trauma - with Tissue Engineering approaches became more and more attractive. In the previously mentioned pathologic entities, tissue damage extends to the subchondral bone and involves two tissues characterized by different intrinsic healing capacities 1. There is an increasing interest in the role of subchondral bone for the pathogenic processes of os...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAN Biotech GmbH</td>
			<td>0401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2210</td>
			<td>1,000 units/10 &mu;g/&mu;l in 0.9% NaCl</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KGaA</td>
			<td>410230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biocoll Separation Sol.</td>
			<td>Biochrom AG</td>
			<td>L6115</td>
			<td>Isotonic solution Density: 1,077 g/ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Invitrogen GmbH</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>DeltaSelectGmBH</td>
			<td>1819340</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>BBraun</td>
			<td>8333A193</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringes (Injekt)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needles (Sterican)</td>
			<td>BBraun</td>
			<td>4657519</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps (blunt/sharp)</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Feather Safety Razor Co</td>
			<td>02.001.30.022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes research</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bone Cutter</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Forma Scientific Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture laminar flow hood Hera Safe</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile saw</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge Megafuge 2.0 R</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Air operated power drill</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TISSUCOL-Kit 1.0 ml Immuno</td>
			<td>Baxter</td>
			<td>2546648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibers (4-0 Monocryl, 4-0 Vicryl)</td>
			<td>Ethicon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
	</tbody>
</table>

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.

The described surgical technique permits a successful isolation and implantation of allogeneic mesenchymal stem cells into an artificial osteochondral defect. The experimental setup resulted in a successful integration of the implant into the surrounding cartilage.
The defect was filled by repair tissue with similar biomechanical properties and similar durability compared to the surrounding cartilage. The fibrin-cell-clot was prepared in vitro on a sterile plate with pre-drilled holes...

Watch this Scientific Journal Video about Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots at JoVE.com

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

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.

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 ...

treatment-osteochondral-defects-rabbit-s-knee-joint-implantation

Research

JoVE Journal

Medicine

17.3K Views.  Klinikum rechts der Isar der Technischen Universität München. 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.

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...