Isolation and characterization of mesenchymal stem cells in orthopaedics and the emergence of compact bone mesenchymal stem cells as a promising surgical adjunct

Table 1 Source and isolation methods of mesenchymal stromal cells

Source	Isolation method	Ref.
Bone marrow	Aspirates cultured and media changed every 3-4 d to select for MSCs	[34]
	Aspirates layered over Ficoll-Paque density-gradient and plates in tissue culture dish. Adherent cells maintained with periodic passaging	[82]
	Bone marrow mononuclear cells seeded from single colony-forming unit fibroblasts and selected for by CD105(+)/CD45(-)	[83]
	Sort bone mononuclear cells based on aldehyde deydrogenase expression (ALDHhighCD45-)	[84]
	Sort based on CD45−/lowCD271+ phenotype following a microbead-based pre-enrichement	[85]
	Layer bone marrow over hyluronic acid followed by centrifugation, collect most superficial layer containing the mononuclear cells	[86]
Compact bone	Trabecular bone fragments rinsed and placed in complete α-MEM/Ham’s F12, confluent monolayers were obtained within 10-20 d	[87-89]
	Bone cell cultures established by treating bone fragments with collagenase in low Ca2+ medium	[9,90,30]
	Compact bone fragments obtained, cultured, and isolated. CB-MSCs then undergo trypsinization to reveal enhanced osteogenic capacity	[43]
Adipose	Place 10-20 mL of washed adipose tissue in 100 mm Petri dish; dissect out yellow tissue; mince tissue finely and place in enzymatic digestion solution; centrifuge and collect pellet for wash; resuspend in complete culture medium	[14,13]
	Wash lipoaspirate with PBS; enzymatically digest using collagenase 1A solution; spin down cells, wash and plate in complete medium	[12]
Bone marrow	Aspirates cultured and media changed every 3-4 d to select for MSCs	[34]
	Aspirates layered over Ficoll-Paque density-gradient and plates in tissue culture dish. Adherent cells maintained with periodic passaging	[82]
	Bone marrow mononuclear cells seeded from single colony-forming unit fibroblasts and selected for by CD105(+)/CD45(-)	[83]
	Sort bone mononuclear cells based on aldehyde deydrogenase expression (ALDHhighCD45-)	[84]
	Sort based on CD45−/lowCD271+ phenotype following a microbead-based pre-enrichement	[85]

MSC: Mesenchymal stromal cell; CB: Compact bone; PBS: Phosphate buffer saline.

Table 2 Potential clinical scenarios for use of mesenchymal stromal cell therapy

Ref.	Animal model/methods	Findings	Conclusion	In vitro/in vivo
Ogulur et al[91]	mCB-MSCs isolated from 6–8 wk old BALB/c mice	mCB-MSCs significantly reduced cellular immune infiltration and presence of goblet cells as well as the thickness of epithelium, smooth muscle layers, and basement membrane in ovalbumin induced chronic asthmatic mice	Inflammation in distal and proximal airways of ovalbumin induced asthmatic mice can be suppressed by use of IV mCB-MSCs	In vivo
Qiao et al[76]	CB-MSCs isolated from C57BL/6 mice administered to 8–10 wk old BALB/c mice	BALB/c mice exposed to 8 Gy TBI and treated with CB-MSCs showed improved survival, body weight, and CFU-GM counts of bone marrow cells coupled with suppressed Th1 immunity with increased Treg percentages and decreased IFN-γ, CXCR3 and CCR5	CB-MSC transplantation post total body irradiation attenuates radiation-induced hematopoietic toxicity and provides immunoprotection	In vivo
Duran et al[92]	Cortical bone–derived stem cells from 12-wk-old EGFP+ transgenic mice	Improved 6 wk survival post MI procedure (50.4% to 76.5%) from saline to CB-MSC therapy. Increased expression of proangiogenic paracrine factors (bFGF and VEGF) and differentiation into infarct zone	Treatment with CB-MSCs post MI leads to enhanced survival, cardiac function, and remodeling	In vivo
Cheng et al[93]	MSCs isolated from compact bone of Tg26 HIV-1 transgenic mice	Transplanted Tg26 HIV-1 MSCs were less effective in protecting renal tubular cells compared to healthy mice MSCs in a cisplatin-induced AKI model due to inferior proliferation and decrease in secretion of protective cytokines	Compact bone MSCs infected with HIV-1 had impaired proliferation, differentiation, and function resulting in less therapeutic potential	In vivo
Yamachika et al[94]	MSCs from compact bone of 5-week-old C57-GFP male mice	Cells cultured in bFGF-conditioned medium demonstrated trilineage differentiation potential even at passage 24 in contrast to leukemia inhibitory factor-conditioned medium	Compact bone MSCs cultured in bFGF-conditioned medium demonstrated bone formation ability in vivo	In vivo
Bakker et al[95]	Tibial reaming debris from adult female sheep	Treatment with reaming debris, similar to iliac crest, revealed larger callus volume with decreased cartilage in the fracture gap, increased bone volume, and improved toughness at 3 wk with greater torsional stiffness at 6 wk	Reaming debris has characteristics similar to iliac crest bone that allow it to be an excellent replacement for enhancing healing of bone defects fixed with an intramedullary nail	In vivo
Guo et al[24]	Murine mesenchymal progenitor cells (muMPCs) isolated from 2-3 wk old C57BL/6 female mice tibia/femur compact bone	Collagenase-digested bone fragments produced muMPCs that inhibited Con A-stimulated splenocyte proliferation and suppressed lymphocyte activation by allogeneic cellular stimuli in vitro. In addition, muMPCs improved survival of allogeneic skin grafts in vivo	Using this protocol allows acquiring of muMPCs with similar properties to marrow counterparts, which allows them to be used in future investigations with mouse models	In vivo
Lim et al[96]	hABMSCs	hABMSCs exposed to low-intensity pulsed ultrasound revealed increased ALP, expression levels of CD29, CD44, COL1, and OCN, and calcium deposition	Treatment with LIPUS could improve the cell viability and osteogenic differentiation of hABMSCs	In vitro
Lim et al[97]	hABMSCs	hABSMSCs treated with extremely low frequency pulsed electromagnetic fields (ELF-PEMFs) revealed 15% increased proliferation at day 5, increased ALP, vinculin, vimentin, and CaM expressions, and enhanced mineralization during osteogenesis	Exposing hABMSCs with ELF-PEMFs could improve and accelerate the process of early cell proliferation mediated osteogenesis	In vitro
Lim et al[98]	hABMSCs harvested from human mandibular alveolar bone	hABSMSCs exposed to LFDSS for 10–60 min/d demonstrated improved viability, proliferation, and mineralization in culture with osteoblasts. ALP activity and gene expression of IBSP, COL-I, OCN, and OPN increased	Proper intensity and exposure time of LFDSS to hABMSCs can improve their differentiation and maturation	In vitro
Soleimani et al[35]	MSCs isolated from 6-8 wk old BALB/c mouse tibial and femoral bone marrow	The protocol states MSCs should be cultured in Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) in a 37 °C–5% CO2 incubator with passage at 2 wk of culture	This protocol allows development of a purified population of MSCs 3 wk after the initiation of culture	In vitro
Dominici et al[44]	Human multipotent MSC	In standard culture, MSC must be plastic-adherent, express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR and demonstrate tridifferentiation in vitro	Standard criteria for MSC characterization, will allow for exchange of more uniform data between researchers	In vitro
Wenisch et al[99]	Mesenchymal stem cells harvested from HRD of 12 adult patients with closed diaphyseal femoral fractures	With neuronal induction, MSCs assumed neuronal morphologies and expressed neuron-specific enolase, beta-III-tubulin, neurofilament-H and HNK-1. Similar to immature neurons, MSCs had features of neuritogenesis and synaptogenesis and lacked electrical signaling	Neuronal induction allowed initiation of the early neuronal differentiation, but exposure to non-neurological stressors led to necrotic alterations	In vitro
Wenisch et al[100]	Mesenchymal stem cells harvested from HRD of 12 adult patients with closed diaphyseal femoral fractures	After multiple passages, HRD-derived cells and MSCs maintained a nondifferentiated phenotype and showed osteogenic and neuronal pathway differentiation ability after induction	Human reaming debris provides a multipotent stem cells which have the ability to grow and proliferate in vitro	In vitro
Tuli et al[90]	Collagenase-treated human trabecular bone chips	Collagenase-treated trabecular bone fragments contain cells that stain positive for CD73, STRO-1, and CD105, and negative for CD34, CD45, and CD144 with tridifferentiation potential	Trabecular bone-derived cells maintain a nondifferentiated phenotype and display tridifferentiation potential with long-term in vitro culture	In vitro

AKI: Acute kidney injury; bFGF: Basic fibroblast growth factor; CBSC: Cortical bone stem cell; CB-MSCs: Compact bone mesenchymal stem cells; CFU-GMC: Colony-forming unit granulocyte/macrophage; LIPUS: Low intensity pulsed ultrasound; MI: Myocardial infarction; TBI: Total body irradiation; VEGF: Vascular endothelial growth factor; HRD: Human reaming debris; hABMSC: Human alveolar bone-derived mesenchymal stem cell; LFDSS: Low fluid dynamic shear stress; ALP: A lkaline phosphatase; HLA: Human leukocyte antigen; HIV: Human immunodeficiency virus.