The restoration of osteochondral defects presents great challenges that have not been fully solved by the current therapies. Therefore, this field continues to expand, bridging the gap between palliative care and defects reconstruction. In the last few years, tissue engineering and regenerative medicine have been offering advanced strategies and some of which have successfully reached clinical application and the market. Beyond the origin and source of cells, the development of culture conditions remains an important step to further clinical applications. Several approaches have been focused on good manufacturing practice (GMP) conditions. The aim is the creation of advanced therapy medicinal products (ATMPs). The up-to-date state of the culture protocols for osteochondral tissue engineering with respect to different cells, growth factors, and biomaterial scaffolds, as well as the strategies employed in clinical trials for the restoration and repair of osteochondral defects, will be the focus of this book chapter.
KeywordsCell culture Clinic Tissue engineering and regenerative medicine Cartilage Bone Good manufacturing practice
Scaffolds employed in osteochondral clinical studies, its administration method, and its corresponding targeted issues
Vascularized iliac bone graft
Osteonecrosis of femoral head
Iliac bone graft
Osteonecrosis of femoral head
Porcine-derived collagen type I/III membrane
Knee cartilage defects
Osteochondral lesions of the talus
Articular chondral lesions
Knee cartilage defects
Demineralized bone matrix (DBM)
Distal tibial fractures
Origin and source of human stem cells applied in bone and cartilage tissue engineering
Adult human peripheral blood CD34+ cells
Synovium-derived mesenchymal stem cells (S-MSCs)
Cortical bone fragment-derived mesenchymal stem cells (CBF-MSCs)
Bone marrow-derived mesenchymal stem cells (BM-MSCs)
Bone and cartilage
Adipose tissue-derived mesenchymal stem cells (AT-MSCs)
Peripheral blood-derived mesenchymal stem cells (PB-MSCs)
Dental pulp-derived mesenchymal stem cells (DP-MSCs)
Adipose-derived stromal vascular fraction (SVF)
Bone marrow-derived mesenchymal stem cells (BM-MSCs)
Embryonic stem cells (ESCs)
TGF-β1 modified human chondrocytes
Umbilical cord blood mesenchymal stem cells (CB-MSCs)
Induced pluripotent stem cells (iPSCs)
49.2 Preclinical Cell Culture Methods
49.2.1 Cell-Based Methods
22.214.171.124 Human Mesenchymal Stem Cell Culture
To restore bone and cartilage defects in clinical applications, some critical issues that are inherently related to the culture conditions must be considered. Human AT-MSCs (also called hASCs) can be obtained from lipoaspirates, which is a less invasive procedure compared to the harvesting of human BM-MSCs, which requires drilling into the bone. hASCs can differentiate into the chondrogenic, osteogenic, adipogenic, myogenic, neurogenic, and hepatogenic lineages . Moreover, stem cell yields are higher from adipose tissue, with 1 g containing an average of 2 × 106 cells with 10% being ASCs . Therefore, adipose tissue represents an abundant and practical source of multipotent stem cells for autologous and allogenic cell transplantation approaches.
Conventional hASC Isolation and Expansion Protocol
Standard methods for isolating and purifying human ASCs.
Ten grams of adipose tissue was harvested, washed in phosphate-buffered saline (PBS) supplemented with 10% penicillin-streptomycin solution (vol/vol).
After washing, the adipose tissue is minced and digested in 15 mL of 37 °C warmed Dulbecco’s modified Eagle’s medium (DMEM)/collagenase type II from Clostridium histolyticum solution (1:1) supplemented with 10% penicillin-streptomycin solution (vol/vol) while shaking at 37 °C and 180 rpm overnight.
After digestion, filter the samples through a 100 μm cell strainer to a new 50 mL Falcon tube, complete to 20 mL with DMEM, and centrifuge 10 min at 800 × g to allow for complete cell/layer separation.
Aspirate the supernatant, resuspend the pellet in 15 mL of PBS to wash the cells, and centrifuge 10 min at 300 × g.
Remove the supernatant and resuspend the pellet in 5–10 mL of DMEM (volume depending on the size of the pellet obtained).
Count the cells using a Neubauer chamber.
The isolated hASCs were seeded in 75 cm2 culture flasks at a density of 5 × 103 cells/cm2, cultured in DMEM supplemented with 10% of fetal bovine serum (FBS) (vol/vol) and 1% of penicillin-streptomycin solution (vol/vol), and incubated at 37 °C with 5% CO2 (Fig. 49.1).
Standard Chondrogenic Differentiation Protocol
hASCs at passage 4 (P4) were cultured in the form of pellets (obtained by centrifugation of 5 × 106 cells at 800 × g) in 15 mL Falcon tube.
The pellets were then cultured in chondrogenic media composed of DMEM/F-12 (with glutamine and sodium pyruvate) or DMEM-high glucose (DMEM-HG) supplemented with 1% FBS (vol/vol), 1% of penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 1% insulin-transferrin-selenium-X (ITS), 50 μg/mL ascorbate-2-phosphate, 40 μg/mL l-proline, 50 ng/mL insulin-like growth factor 1 (IGF-1), and 10 ng/mL TGF-β1 for 21 days.
The cells were incubated at 37 °C with 5% CO2.
The medium was replaced every 3 days.
After the 21 days, the pellets were collected. Some of them were fixed in 4% neutral buffered formalin and used for chondrogenic histological analysis (Alcian blue, toluidine blue, and safranin O staining). The remaining pellets were used to evaluate the gene expression levels of the chondrogenic markers, collagen II, aggrecan (ACA), and Sox9 by quantitative reverse transcription PCR (RT-qPCR) [45, 46, 47].
Standard Osteogenic Differentiation Protocol
hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2.
The cells were cultured in osteogenic media composed of DMEM or α-MEM supplemented with 1% FBS (vol/vol), 1% of penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 10 mM β-glycerol phosphate, and 5 mg/mL ascorbate-2-phosphate for 21 days.
The cells were incubated at 37 °C with 5% CO2.
The medium was replaced every 3 days.
After the 21 days, some of the wells were fixed in 4% neutral buffered formalin and used for osteogenic histological analysis (alizarin red staining). The remaining wells were used to evaluate the gene expression levels of the osteogenic markers, alkaline phosphatase (ALP), osteocalcin (OSC), osteopontin (OPN), and Runx2 by quantitative reverse transcription PCR (RT-qPCR) [45, 46, 47].
To be able to use cells clinically, it is required to define good manufacturing practice (GMP) methods to isolate as well as to culture them in a well-defined medium under GMP conditions. A number of different companies together with research laboratories started to develop automated closed devices to standardize parts or even the whole cell isolation process. Oberbauer summarized a survey of currently patented, published, or commercially available enzymatic and nonenzymatic adipose tissue-derived cell isolation systems .
Several factors can influence clinical cell culturing, such as cell culture media, medium supplements, cell density, surface coating, detachment enzymes, oxygen conditions, and cell plating density. Nevertheless, the medium used for expansion of the cells represents the most crucial factor . Some concerns do exist with the use of FBS for clinical applications, due to (a) the possibility that FBS contains unsafe contaminants such as prions, viral, and zoonotic agents and (b) its inconsistency from batch to batch.
Furthermore, when hASCs are cultured in a medium comprising animal proteins, a large amount of these proteins is retained in the cytoplasm, which can stimulate an immunologic reaction when transplanted in vivo. Therefore, efforts have been made by several international research groups to develop (a) alternative media either by replacing FBS with human-sourced supplements (such as platelet-rich plasma or human serum) or by identifying defined serum-free formulations consisting of key growth/attachment factors and (b) controlled bioreactor protocols.
126.96.36.199 “Humanized” hASC Culture Protocols
The present chapter will focus on the developed alternatives which will guarantee the safe, economical, and ethical practice of biomedical research, through the elimination of many shortcomings of FBS. These alternatives are based on the use of medium supplemented with human platelet-rich plasma (PRP) or human autoserum and on culture under animal-/xeno-free media conditions.
Platelet-Rich Plasma Supplement
Over the last two decades, human platelet-rich plasma (PRP), an autologous derivative of whole blood, has been widely used as therapy for orthopedic injuries . PRP is rich in growth factors including transforming growth factor β1 (TGF-β1), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) .
Platelet-Rich Plasma Preparation
Human PRP was obtained by venipuncture in acid citrate dextrose (ACD) tubes.
Briefly, 10 mL of venous blood was drawn from the antecubital vein in sterile vacuum tube containing 1 mL citrate phosphate dextrose adenine (CPDA) as anticoagulant and centrifuged at 250 × g for 15 min.
After the first centrifugation, two layers were seen clearly. The superior yellow layer was consisting of platelet-rich (PRP) and platelet-poor (PPP) plasma, and lower red layer was consisting of erythrocytes and leukocytes.
Then the complete upper yellow layer was transferred into plain vacuum tube.
After the second centrifugation at 200 × g for 15 min, approximately 2 mL was PRP at the bottom of the vacuum tube and the upper rest was plasma PPP fraction.
The PRP was activated with 10–20% CaCl2, thrombin solution.
Platelet-Rich Plasma Culture Medium Supplementation
Human ASCs will be isolated under GMP conditions.
For basal expansion/growth, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Then, cells were cultured in DMEM supplemented with 1% penicillin-streptomycin solution and 20% PRP (vol/vol), prepared and activated as previously described. This concentration of PRP offers the optimal condition for cell growth with increased proliferation compared to FBS supplementation . The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
For chondrogenic differentiation, hASCs at passage 4 (P4) were cultured in the form of pellets (obtained by centrifugation of 5 × 106 cells at 800 × g) in 15 mL Falcon tube. Then, cells were cultured in chondrogenic media composed of DMEM/F-12 (with glutamine and sodium pyruvate) or DMEM-high glucose (DMEM-HG) supplemented with 20% PRP (vol/vol), 1% of penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 1% insulin-transferrin-selenium-X (ITS), 50 μg/mL ascorbate-2-phosphate, 40 μg/mL l-proline, 50 ng/mL insulin-like growth factor 1 (IGF-1), and 10 ng/mL TGF-β1 for 21 days. The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
For osteogenic differentiation, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Then, cells were cultured in DMEM supplemented with 1% penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 10 mM β-glycerol phosphate, 5 mg/mL ascorbate-2-phosphate, and 20% of PRP (vol/vol) for 21 days . The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
Besides, the clinical advantages of PRP such as (a) its non-stimulative and bioactivity-supporting role, (b) its outstanding cost to benefit ratio, (c) its preparation that does not require expensive devices, and (d) its low risk for infection, the careful preparation, and quality control of PRP, in combination with the rigorous evaluation of recipient implantation sites, will lead to improved clinical outcomes for patients .
Human Autoserum-Based Supplement
Autologous human serum was investigated as a substitute for fetal bovine serum in the ex vivo expansion medium to avoid the transmission of dangerous pathogens/contaminants during clinical reconstruction procedures.
Human Autoserum Preparation
Human serum was derived from whole blood donations.
Afterward, the blood was centrifuged at 1.800 × g for 15 min at 4 °C.
Serum was collected and filtered through 0.2 μm membrane, and sterile aliquots were stored at −20 °C.
Human Autoserum (hAS) Culture Medium Supplementation
Human ASCs will be isolated under GMP conditions.
For basal expansion/growth, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Cells were cultured in DMEM supplemented with 1% penicillin-streptomycin solution (vol/vol) and 10% hAS (vol/vol). The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days .
For chondrogenic differentiation, hASCs at passage 4 (P4) were cultured in the form of pellets (obtained by centrifugation of 5 × 106 cells at 800 × g) in 15 mL Falcon tube. Then, cells were cultured in chondrogenic media composed of DMEM/F-12 (with glutamine and sodium pyruvate) or DMEM-high glucose (DMEM-HG) supplemented with 10% hAS (vol/vol), 1% of penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 1% insulin-transferrin-selenium-X (ITS), 50 μg/mL ascorbate-2-phosphate, 40 μg/mL l-proline, 50 ng/mL insulin-like growth factor 1 (IGF-1), and 10 ng/mL TGF-β1 for 21 days. The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
For osteogenic differentiation, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Then, cells were cultured in DMEM supplemented with 1% penicillin-streptomycin solution (vol/vol), 100 nM dexamethasone, 10 mM β-glycerol phosphate, 5 mg/mL ascorbate-2-phosphate, and 10% of hAS (vol/vol) for 21 days . The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
Nevertheless, the use of hAS showed arguments such as high costs and the high likelihood of factors in the blood of donors, which may dominantly impact on hASC growth, greatly promoting the development of serum-free, chemically defined media.
Serum-Free/Xeno-Free Medium-Based Protocol
Alternatives for FBS have been previously described, such as platelet-derived supplements and human autoserum (hAS). However, restrictions related to the use of serum or its components exist, such as batch variation with serum-supplemented media performance, affecting proliferation rate and differentiation potential. Therefore, defined serum-free (SF)/xeno-free (XF) media formulations have been developed in order to enhance significantly the safety and quality of the transplanted stem cells . These chemically defined media presented some advantages, namely, their precise chemical composition and the absence of xenogeneic contaminants (i.e., microorganisms). Nevertheless, the optimal medium composition mostly depends on the requests of individual experimental conditions and may vary largely between and within cell types.
Human ASCs will be isolated under GMP conditions.
For basal expansion/growth, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Cells were cultured in SF/XF media supplemented with 1% penicillin-streptomycin solution (vol/vol). Depending on the SF/XS medium manufacturer’s guidelines, other supplements might be added. The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days.
For chondrogenic differentiation, hASCs at passage 4 (P4) were cultured in the form of pellets (obtained by centrifugation of 5 × 106 cells at 800 × g) in 15 mL Falcon tube. Then, cells were cultured in xeno-free chondrogenic medium supplemented with 1% of penicillin-streptomycin solution (vol/vol) for 21 days. The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days [63, 64].
For osteogenic differentiation, hASCs at passage 4 (P4) were seeded in 24-well TCPS plate at a density of 15 × 103 cells/cm2. Then, cells were cultured in xeno-free osteogenic medium supplemented with 1% penicillin-streptomycin solution (vol/vol) for 21 days. The cells were incubated at 37 °C with 5% CO2. The medium was replaced every 3 days [63, 64].
49.2.2 3D-Based Protocols
Three-dimensional (3D) matrices have been studied to improve cell expansion through the delivery of an active surface for cells to attach and to proliferate. This method provides main advantages such as: (a) an increased surface to volume ratio, thus allowing the growth of cells within a confined volume space; (b) for adherent-dependent cells, the surface properties can be functionalized to improve attachment and proliferation; and (c) 3D surfaces increase the possibilities of providing a microenvironment that can control cell behavior and function by engineered biomaterials and cell niches, naturally seen in in vivo physiology. The following four methods are presently applied for adherent cell-based expansion for clinical trials and commercial production.
Current approaches for bone and cartilage repair are centered on the use of scaffolds providing a suitable three-dimensional (3D) environment supporting the growth of a repair tissue. These 3D structures are often critical, both in vitro and in vivo, to summarizing the in vivo milieu and allowing cells to modulate their own microenvironment. The ideal scaffolds for bone and cartilage tissue engineering must be based on the following basic requirements: porous, biocompatible, biodegradable, and appropriate for cell attachment, proliferation, and differentiation. Several biomaterials have been studied for cartilage and bone tissue engineering [65, 66].
Human ASCs at passage 4 (P4) were expanded in xeno-free media supplemented with 1% penicillin-streptomycin solution (vol/vol) until sub-confluency.
When reaching 80% confluence, cells were trypsinized using TrypLE Express solution.
Add the required amount of GG-MA with 9 mL of warm, sterile distillated water.
Allow it to dissolve in 37 °C water bath.
Prepare a cell suspension of 3 × 106 cell in 300 μL of cell culture xeno-free media supplemented with 1% penicillin-streptomycin solution (vol/vol).
Add 2.7 mL of GG-MA hydrogel to the cell suspension and quickly mix by pipetting up and down (hydrogel concentration is 2% w/V, cell density is 1 × 106 cell/mL).
Pipette 50 μL of cellular hydrogel to each well.
Cover the hydrogel with 500 μL of basal culture xeno-free medium or xeno-free chondrogenic medium, both supplemented with 1% penicillin-streptomycin solution (vol/vol) for 21 days.
Incubate the tridimensional culture at 37 °C with 5% CO2. The medium was replaced every 3 days.
After the time of incubation, this new ATMP will be administrated into the cartilage defect in vivo .
Sterile scaffolds should be pretreated to prevent air bubble formation in the pores, which can inhibit cell adhesion, through the use of a 20 mL sterile syringe with an attached 21G sterile needle.
Before the cell seeding, the scaffolds were hydrated in culture medium overnight in the CO2 incubator at 37 °C.
Afterward, the scaffolds were removed from the medium and placed into a 48-well suspension cell culture plate.
Seed the hASCs at passage 4 (P4) onto the surface of the scaffolds at different cell densities (e.g., 5 × 102 cells/scaffold, 1 × 103 cells/scaffold, 5 × 103 cells/scaffold, and 1 × 104 cells/scaffold).
Then the scaffolds with cells were kept in the CO2 incubator at 37 °C.
After 3h, the constructs were transferred to a new 48-well suspension culture plate, and each construct was supplemented with 1 mL of culture medium.
Incubate the tridimensional culture at 37 °C with 5% CO2. The medium was replaced every 3 days.
Large-scale hASC manufacturing should rely on effective and economical GMP-compliant processes that were able to strongly generate cells with well-defined characteristics in quantities that meet clinical demands. These procedures should offer consistent optimal growth conditions for hASC, complete monitoring, and control of culture conditions, while presenting easy scalability. To encounter these supplies, stirred bioreactors offer substantial advantages over static systems. In a static cell culture condition, the environment is continuously changing through the accumulation of metabolites, nutrient depletion from the culture medium, and the resultant changes in pH. This is evidently not representative with the situation in vivo, where cells are continuously in equilibrium between the constant elimination of products and supply of nutrients. In tissue engineering (TE), bioreactors might be used for different purposes: (a) culture/growth of cells (generally in suspension) prior seeding onto the scaffold biomaterial and (b) to achieve specific physical stimulation of the TE construct, in order to support cell/tissue differentiation. The advantages of bioreactors on cell expansion and tissue maturation have been clearly demonstrated .
Therefore, stirred bioreactors allow a more homogeneous culture environment and guarantee monitoring and control of key culture factors (e.g., pH, temperature, oxygen contain), while reducing culture handling and costs [72, 73].
Use of Bellco spinner flasks with a working volume of 80 mL, equipped with 90° paddles (normal paddles) and a magnetic stir bar.
The initial hASC density used was 5 × 104 cells/mL.
Plastic microcarriers were coated with a CELLstart CTS solution (diluted 1:100 in PBS with Ca2+ and Mg2+) for 2 h at 37 °C, with an intermittent agitation (1 min at 300 rpm, 10 min non-agitated) using a Thermomixer comfort, and afterward equilibrated in pre-warmed MesenPRO RS/StemPro MSC SFM XenoFree medium.
hASC, previously expanded under xeno-free static conditions for two passages, was seeded on 20 g/L of pre-coated plastic microcarriers in 15 mL of the respective medium for 30 min, at 37 °C and 5% CO2, with gentle agitation every 5 min.
Then, pre-warmed medium was added until reaching half of the final volume, and the cell suspension was transferred to the spinner flask and submitted to an intermittent agitation regimen (15 min at 25 rpm followed by 2 h non-agitated) during 24 h.
After the initial 24 h, agitation was set at 40 rpm.
After 3 days of culturing, 25% of the medium volume was renewed daily.
Moreover, hASC demonstrated efficient expansion using a controlled stirred-tank bioreactors .
After 4 days in the spinner flasks, the microcarrier-cell suspension was transferred to a 1300 mL stirred tank bioreactor equipped with a 3-blade pitched impeller (blades pitched 4° to vertical) and dissolved oxygen and pH probes.
The addition of CO2 was made through gentle sparging from the base of the reactor, and 1.0 M of NaHCO3 was used to maintain pH of culture.
Xeno-free culture medium was added to reach the working volume of 800 mL.
The culture parameters were set to pH 7.2, continuous agitation at 60 rpm, and aeration. According to the experimental setup, dissolved oxygen was set to 9 or 20% of air saturation.
188.8.131.52 Bioreactors Available for Cartilage TE
The improvement of cartilage engineering through the use of bioreactors is well demonstrated [76, 77, 78]. To overcome limitations resulting from the mass transport of nutrients into the core of large scaffolds that hereafter affected the extent to which such constructs could mimic native cartilage, more complex bioreactors have been designed i.e., spinner flasks, rotating wall vessel bioreactors, and flow perfusion bioreactors . Recently a study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation .
184.108.40.206 Bioreactors Available for Bone TE
Ex vivo TE approaches for de novo generation of bone tissue enclose the combined use of autologous bone-forming cells (hASCs) and three-dimensional scaffold materials serving as structural support for the cells [80, 81]. As for cartilage TE, several complex bioreactors have been developed, i.e., rotating wall vessel bioreactor, spinner flask bioreactor, perfusion bioreactor, as well as compression bioreactors and combined systems [82, 83].
49.3 Clinical Cell Culture Methods
49.3.1 Cell-Based Methods
220.127.116.11 Autologous Cells
Standard techniques such as marrow stimulation, promoted by subchondral drilling or microfracture, and autologous grafts to repair chondral and osteochondral defects present some problems. Recent advances in orthopedics have prompt the use of stem cell transplant treatment to solve some issues that arise with traditional approaches. In fact, bone can reform when damage, but it may develop fibrous cartilage, which can be avoided by using cells with osteogenic potential. Stem cells exist in almost all tissues and can differentiate into specialized cells, such as bone and cartilage cells when submitted to specific cues. Once transplanted, these cells adapt to the surrounding environment and differentiate upon niche factor stimulation. This way, they help in regenerating damage areas. For example, in the case of autologous grafts, cells can be helpful in promoting the union between graft and damage area. Nevertheless, cell culture methods should be adapted for clinical applications as described along this section.
The most used cells are bone marrow-derived mesenchymal stem cells (BM-MSCs) [22, 84, 85]. BM-MSCs are usually obtained from bone marrow harvested from patient’s own iliac crest, but different isolation protocols can be followed prior to the implantation. For example, cells can be separated using dextran sedimentation , Ficoll density centrifugation media , Percoll density centrifugation media , MarrowStim Concentration System® , and ART BMC system .
The culture step is common practice to generate MSCs because the marrow aspirate contains a mixture of different red blood cells, platelets, and leukocytes (a majority consists mostly of neutrophils, lymphocytes, and monocytes). Additionally, MSCs are cultured in a class B clean room environment. During the procedure MSC cultures should be subjected to several quality control tests, such as mycoplasma and microbial contamination (aerobic and anaerobic bacteria, as well as fungus). To confirm the identity of stem cells, they can be further characterized for MSC markers in accordance with the recommendations of the International Society for Cell Therapy .
Harvest bone marrow blood from iliac crests using a syringe that had been flushed with heparin.
Store the aspirated bone marrow blood in a sterile plastic bag containing an anticoagulant solution (citric acid, sodium citrate, and dextrose).
Filter and wash the aspirated bone marrow to remove fat and clot debris.
Isolate BM-MSCs by a density centrifugation method.
After centrifugation, isolate and remove the red blood cells (the nonnucleated cells) and the plasma.
Yield and place BM-MSCs in syringes for injection.
Inject BM-MSCs into the lesion slowly.
After separation, BM-MSCs are injected inside the graft itself . To avoid leakage allograft bone plug can be used as done by Tabatabaee et al.  or sealed with bone wax as done by . In a different approach, BM-MSCs can be combined with autologous peripheral blood mixed with an internal bone graft to serve as a natural scaffold as done by Weel et al. .
Stem cells obtained from adipose tissue have emerged as a very attractive source. In fact, adipose tissue is an abundant, accessible, and rich source of adult stem cells with multipotent properties. Commonly, adipose tissue is obtained during abdominal liposuctions [25, 91], but it can also be harvested from infrapatellar fat pad during an arthroscopic surgery . Then, the aspirate is processed in a sterile environment in a Biological Safety Cabinet (BSC) Class II, using strict aseptic techniques at a GMP facility.
Aspirate adipose tissue.
Mix adipose tissue with collagenase solution and incubate at 37 °C for 45 min.
Stop enzymatic digestion by adding complete culture medium.
After homogenization, pass the digested suspension through sterile filters.
Centrifuge cells at room temperature for 10 min at 600 × g.
Discard the supernatant and resuspend in complete culture medium.
Remove an aliquot for quality control (e.g., cell count, viability, phenotyping, and sterility).
Seed cells in complete culture medium, at 37 °C in an atmosphere saturated with moisture and 5% CO2.
After 24 h incubation, wash adherent cells to remove the nonadherent cells.
Then, harvest cells with the use of irradiated trypsin solution.
Remove an aliquot of the cell suspension for quality control as above.
Inject ASC at lesion site.
Furthermore, other cells were studied to repair chondral and osteochondral defects. Autologous chondrocyte implantation (ACI) is a conventional method for treatment of cartilage defects of the knee joint [15, 93]. In this case, chondrocytes are isolated from cartilage biopsy obtained under arthroscopy. Usually, after injection of chondrocytes, there is a need to seal the defect with fibrin glue .
Harvest cartilage biopsy during an arthroscopy.
Release cells from the fragment through enzymatic digestion.
Culture cells in serum taken from the patient’s blood at the time of surgery.
Finally, inject the expanded chondrocytes.
The procedures frequently performed to treat intervertebral disk degeneration are microscopic diskectomy or spinal fusion. Although they are clinically effective, they do not preserve the function of intervertebral disk and enhance the mechanical loads that the disks are submitted to. For so, new therapies have been pursued as the use of nucleus pulposus cells .
Nucleus Pulposus Cell Isolation
Collect tissue from degenerated intervertebral disk.
Isolate nucleus pulposus under a dissecting microscope.
Transfer nucleus pulposus tissue to a culture dish and clean the attached blood with a saline solution.
Place the tissue into a conical tube and weigh.
Transfer it back to a culture dish.
Mince the tissue using a sterile scalpel to obtain small pieces.
Transfer the minced tissue again into a conical tube using saline solution.
Centrifuge the minced tissue at 400 × g for 5 min.
Keep the supernatant for sterility test on bacillus and mycoplasma.
Resuspend the tissue with culture medium with patient’s blood serum and trypsin for 1 h at 37 °C.
Centrifuge again at 400 × g for 5 min and remove the supernatant.
Resuspend the tissue with type I collagenase in culture medium with patient’s blood serum for 2 h at 37 °C.
Centrifuge at 400 × g for 5 min and remove the supernatant.
Suspend the debris in culture medium with patient’s blood serum and pass through a cell strainer to isolate the nucleus pulposus cells.
Culture a monolayer of nucleus pulposus cells at 37 °C in 5% CO2 inside the incubator.
After 4 days, to activate the nucleus pulposus cells, co-culture them with BM-MSC extracted from the patient.
Use a cell scraper to obtain nucleus pulposus cells to administrate to the patient.
Centrifuge the collect cells at 1800 rpm for 5 min.
Retain the supernatant medium for sterility testing for bacillus, virus, mycoplasma, and endotoxin.
Wash twice the nucleus pulposus cells with saline solution and suspend with saline solution.
Retain a small volume of the final suspension for sterility testing for bacillus, virus, and mycoplasma.
Transplant the cells into the degenerated disk.
Cell cultures are usually performed using humanized culture conditions, i.e., platelet-rich plasma, autoserum, or animal-/xeno-free medium to avoid any immune reactions [21, 24, 94]. Nevertheless, in some studies cells were cultured with bovine serum, which was removed and substituted by autologous serum at the moment of implantation . Additionally, it allows the high concentration of autologous growth factors and bioactive molecules, which can influence cell behavior.
Platelet-Rich Plasma (PRP) Preparation
Collect venous blood in a bag containing sodium citrate.
Centrifuge the sample at 1800 rpm for 15 min to separate the erythrocytes and then at 3500 rpm for 10 min to concentrate the platelets.
Send some PRP for analysis of platelet concentration and quality testing (bacteriologic tests).
Before injection, add calcium chloride to the PRP unit to activate the platelets.
Collect peripheral blood without anticoagulant.
In the safety cabinet, allow the peripheral blood to stand for 1 h at room temperature, to achieve a complete coagulation reaction.
After coagulation, centrifuge the blood sample at 1100 × g for 10 min and separate the supernatant of the serum.
Retain a portion of the serum for sterility testing.
18.104.22.168 Allogenic Cells
A different approach has been pursue to enhance cartilage regeneration. A cell-mediated cytokine gene therapy was designed to promote cartilage tissue regeneration, TissueGene-C. For that, chondrocytes have been virally transduced with TGF-β1 [34, 35, 95, 96]. Studies have suggested that TGF-β1 stimulates proteoglycan synthesis in chondrocytes and the growth of articular chondrocytes , which, associated with its anti-inflammatory and immune suppressive properties, has prompt its use for osteochondral approaches.
TGF-β1-Modified Human Chondrocytes(TissueGene-C)
TissueGene-C is supplied in separate vials containing non-modified and modified chondrocytes.
Prior to dosing, wash modified cells with culture medium and resuspend in culture medium.
Irradiate the resuspended modified cells with 15 Gy radiation, to render replication incompetent.
Wash the non-modified cells with culture medium and resuspend in culture medium.
Mix the two types of cell at a 3:1 ratio (non-modified: modified) and load the final mixture into a syringe for injection.
Perform a Gram stain on the final prepared dose for immediate evidence of sterility.
At the same time, test an aliquot of the final preparation for sterility and endotoxin.
To avoid shearing of the cells, the injection should be performed slowly.
49.3.2 Scaffold-Based Methods
Advancements in our understanding of the biology of cartilage and bone healing prompt to the development of new strategies for tissue regeneration. Most of those aim at facilitating tissue regeneration by using scaffolds and cells. Scaffolds can be provided by various types of matrices, such as collagen or even bone grafts [1, 3, 9]. Although allografts have been widely used, they usually failed. On contrary, the addition of cells enhances its success rate . It is important to mention that allografts should irradiate before its use to avoid immune reactions. Recently, new studies had shown that besides the addition of cells, the use of vascularized bone grafts could improve even more the success of autograft-based strategies [1, 2]. Different approaches were followed to progress the success of vascularized bone grafts. In one study, BM-MSCs were mixed with β-tricalcium phosphate granules to induce osteogenic differentiation and transplanted the necrotic area with the vascularized iliac graft . Zhao et al. co-culture iliac bone chips with BM-MSC, both harvested from the patient’s body and transplanted at the lesion area . Then, the vascularized iliac bone graft was inserted into the affected area. In a different approach, Kuroda et al. used a hematopoietic/endothelial progenitor cell-enriched population, CD34+ cells, derived from the bone marrow to promote a similar effect, of a vascularized bone graft . For that, CD34+ cells were dissolved in atelocollagen gel for retaining the cells at the transplanted site. Then, the iliac bone graft was obtained and implanted at the lesion site, where the mixture was locally administered using a syringe.
For treatment of chondral defects, autologous chondrocytes can be implanted and covered with a collagen I/III membrane (ACI)  or seeded onto a purified, resorbable, porcine-derived collagen type I/III membrane [5, 6, 7]. The last method is termed matrix-assisted chondrocyte implantation (MACI) and it is associated with high failure rates. Upon implantation, MACI is usually implanted with the cells facing the bone and secured in place using a thin layer of fibrin glue sealant, while in ACI method the collagen membrane is sutured into position using absorbable sutures.
Different scaffolds were motif of interest as collagen- or hyaluronan-based scaffolds [9, 10]. These scaffolds were loaded with BM-MSC or chondrocytes and placed at the lesion site. Nevertheless, less invasive procedure was pursued through the use of injectable matrices (e.g., sodium hyaluronate, fibrin glue, or demineralized bone matrix) [11, 12, 13]. Regarding the last matrices, stem cells were suspended within each matrix and injected into the lesion.
For these culture methods, humanized protocols were used and cells were isolated accordingly with aforementioned protocols.
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