Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection

Table 1 Summary of the clinical trials involving treatment of the bone defects using adipose-derived stem cells

Bone defect treated	Study duration and length of follow up	n	Intervention	ADSCs source	ADSCs number	Outcome	Ref.
Avascular necrosis of hip, osteoarthritis of hip/knee/ankle, spinal disc herniation	2009-2012, 30 mo	91	Intraarticular injection of SVF with PRP	Autologous SVF from abdominal tumescent liposuction	10 mL of SVF	No evidence of neoplasm, no serious adverse events, common adverse events (swelling of injected joints, tenosynovitis, and tendonitis) were either successfully managed or self-limited, established safety of ADSCs	Pak et al[29]
Upper arm fracture in elderly patients (62-84 yr)	2012-2014, 6 mo	8	SVF seeded porous silicated-hydroxyapatite microgranules with fibrin hydrogel implant	Autologous SVF from abdominal tumescent liposuction	800 microliters of SVF	Evidence of osteogenesis at graft site; circumstantial evidence for direct contribution of SVF cells to fracture healing	Saxer et al[30]
Large cranial defect	2008-2010, 12 mo	4	ADSCs-seeded β-tricalcium phosphate implant	Autologous ADSC from abdominal subcutaneous liposuction	15 × 106 cells	Noted equivalence between newly generated tissue and native bone	Thesleff et al[31]
Large cranial defect	2008-2016, approximately 7 yr	5	ADSCs-seeded β-tricalcium phosphate implant	Autologous ADSC from abdominal subcutaneous liposuction	15 × 106 cells	This study was long term follow up of Thesleff et al[31]; unsatisfactory long-term outcome with significant resorption	Thesleff et al[32]
Cranio-maxillofacial hard-tissue defects	2012-2014, up to 52 mo	13	ADSCs-seeded bioactive glass or β-tricalcium phosphate scaffolds, at times with recombinant hBMP-2	Autologous ADSC from anterior abdominal wall liposuction	Up to 160 × 106 cells	Majority of patients achieved satisfactory clinical and radiographic results; three experienced significant resorptions of the ADSCs graft	Sándor et al[33]
Long bone nonunion from bone tumor resection or pseudoarthrosis	2012-2014, 39 mo	6	ADSCs seeded decellularized bone matrix	Subcutaneous autologous ADSCs	Up to 200 × 106 cells	50% of the patients achieved bone regeneration and union	Dufrane et al[34]
Maxillary sinus floor elevation	2009-2015, 36 mo	10	SVF seeded β- tricalcium phosphate implant	Autologous SVF from abdominal tumescent lipo-aspiration	20 × 106 cells	Experimental group exhibited significantly more bone healing compared to control	Prins et al[35]
Alveolar cleft osteoplasty	2015-2016, 6 mo	10	Lateral ramus cortical bone plate with ADSCs-mounted natural bovine bone mineral	Autologous ADSCs from buccal fat pad	1.0 × 106	No significant different in bone regeneration found between experimental group and controls	Khojasteh et al[36]
Mandibular fracture	2010-2015, 12 wk	20	Direct application of ADSCs	Autologous ADSCs	Unreported	Significantly more osteogenesis in ADSCs-treated group compared to control	Castillo-Cardiel et al[37]
Nonunion following subtalar arthrodesis	2010-2016, 24 mo	140	ADSC-seeded partially demineralized bone matrix	Allograft ADSCs	Unreported	Inferior bone union rate in ADSCs treated group compared to autograft; equivalent clinical evaluations	Myerson et al[38]

ADSCs: Adipose-derived stem cells; SVF: Stromal Vascular Fraction; PRP: Platelet-rich plasma.

Table 2 Summary of the preclinical studies involving bone regeneration induced by transplantation of adipose-derived stem cells

Animal model	Scaffold used	ADSCs per implant	Time frame	Defect healing outcomes	Ref.
-Beagle Dogs; -Unilateral radial segmental defect-10 mm	β-TCP/poly l-lactide-co-glycolide-co-ε-caprolactone composite scaffold	1 × 106 canine ADSCs	20 wk	33.90 ± 4.31	Kang et al[56]
-Wistar albino rats; -Middle zygomatic arch defect; -3 mm wide	No scaffold	Rat inguinal fat pad derived SVF	20 wk	The average new bone growth in the experimental group was 1.1 mm, significantly higher than control	Toplu et al[57]
Group 1: Pre-differentiated ADSCs
-New Zealand white rabbits; -Mid-diaphysis of left ulna; -20 mm long	Porous polylactic glycolic acid scaffold	1 × 106 rabbit SVF cells	8 wk	Approximately 55%	Kim et al[58]
-Beagle dogs; -Parietal bone; -20 mm × 20 mm full-thickness defect	Coral scaffold	60 × 106 of canine ADSCs	24 wk	84.19 ± 6.45	Cui et al[59]
-Lewis rats; -Calvarial defect -8 mm wide	Polylactic scaffold	0.1 × 106 rat ADSCs	8 wk	Coculture of endothelial- and osteoblast-induced ADSC showed no significant improvement over undifferentiated cells	Shah et al[60]
-Lewis rats; -Calvarial defect; -8 mm wide	Poly (D,L-Lactide) scaffold	0.1 × 106 rat ADSCs	8 wk	Osteogenic-induced ADSC generated 0.91 ± 0.65 mm3 new bone, significantly higher than endothelial-induced ADSC	Sahar et al[61]
Group 2: FGF, VEGF, PDGF, and ADSCs
-Osterix‐mCherry reporter mice; -Closed transverse diaphysis fractures of the right femur	No scaffold	0.3 × 106 wild-type mice ADSCs	35 d	The experimental group induced significantly larger mineralized surface and bone callus compared to cell-free and non-transduced controls.	Zhang et al[62]
-Balb/c nude mice; -Parietal bone defect; -4 mm wide	Whitlockite‐reinforced gelatin/heparin cryogels	1 × 106 human ADSCs	8 wk	> 16%	Kim et al[63]
-CD1 nude mice; -Parietal bone defect; -4 mm wide	Coral scaffold	1.5 × 106 human ADSCs	8 wk	95.40%	Behr et al[64]
-Sprague Dawley rats; -Distal femoral cancellous bone -3.5 mm wide and 5 mm deep defect	Trimodal mesoporous bioactive glass scaffold	20 × 106 cell/mL until saturation; rat ADSCs	8 wk	14.25 ± 3.57	Du et al[65]
-Nu/Nu J mice; -Parietal bone; -4 mm wide	Polycaprolactone - fibrin scaffold containing heparin-conjugated decellularized bone	0.2 × 106 human ADSCs	12 wk	The experimental group induced a significantly larger new bone volume compared to the control without PDGF	Rindone et al[66]
Group 3: BMP and ADSCs
-Sprague Dawley rats; -Full-thickness parietal bone defect -5 mm wide	Polylactic glycolic acid scaffold	0.0025 × 106 human ADSCs	8 wk	33.3 ± 29.0	Park et al[67]
-Chinese white rabbits; -Full-thickness calvarial defects; -8 mm	Fibrin gel matrix	3 × 106 rabbit ADSCs	12 wk	Approximately 48	Lin et al[68]
-Japanese white rabbits; -Segmental radial defect; -15 mm	Nano-hydroxyapatite/recombinant human-like collagen/poly (lactic acid) scaffold	2 × 106 cells/ml; rabbit ADSCs	12 wk	97.25 ± 2.06	Hao et al[69]
-Taiwan Lee-Sung minipigs; -Mid-shaft left femur defect; -30 mm long	Apatite coated poly (L-lactide-co-glycolide) scaffolds	100 × 106 cells/animal; minipig ADSCs	12 wk	Experimental group’s new bone formation showed equivalent density and volume compared to native bone and is significantly better than non-transduced control	Lin et al[70]
-CD-1 nude mice; -Full-thickness parietal bone defect -3 mm wide	Porous poly(lactic-co- glycolic acid) scaffold	3 × 106 cells/mL; ADSC from C57BL/6 mouse	6 wk	77%	Fan et al[71]
-Nude mice; -Parietal bone defect; -4 mm wide	Polylactic glycolic acid scaffold	5 × 105 human ADSCs	12 wk	83%	Li et al[72]
-Nude mice; -Subcutaneous implantation	Porous poly(lactic-co- glycolic acid) scaffold	0.01 × 106 rat ADSCs	4 wk	Transduced ADSC construct induced more bone and vessel formation compared to cell-free and non-transduced control	Weimin et al[73]
-CD‐1 nude mice; -Right parietal bone defect; -4 mm wide	Polylactic glycolic acid scaffold	0.15 × 106 human ADSCs	6 wk	Up to 100%	Levi et al[74]
-Athymic nude rat; -Mandible defect; -5 × 5 mm	Chitosan/chondroitin sulfate scaffold	0.25 × 106 ADSCs from C57BL/6 mouse	8 wk	Approximately 43%	Fan et al[75]
Group 4: Genetically manipulated ADSCs
-BALB/c nude mice; -Subcutaneous implantation	β-tricalcium phosphate scaffold	2 × 106 human ADSCs	8 wk	Approximately 30%	Wang et al[76]
-Sprague Dawley rats; -Calvarial defect; -8 mm wide and 1 mm thick	Poly (sebacoyl diglyceride) scaffold	Rat ADSCs	8 wk	50.53 ± 4.45	Xie et al[77]
Group 5: Engineered scaffolds and ADSCs
-C57BL6/J mice; -Mid femur defect; -2 mm	Strontium-substituted hydroxyapatite poly (γ-benzyl-l-glutamate) scaffold	5 × 106 C57BL6/J mice ADSCs	8 wk	Approximately 38%	Gao et al[78]
-Sprague Dawley rats; -Full-thickness femur defect; -4 mm wide	NaB/polylactic glycolic acid scaffold	1 × 106 rat ADSCs	4 wk	ADSC-seeded poly lactic glycolic acid scaffold with 0.05% NaB induced the highest bone density, compared to cell-free control and other concentration of NaB	Doğan et al[79]
-Balb/c nude mice; -Cranium defect; -4 mm wide	SiRNA lipidoid nanoparticle immobilized on polydopamine coated PLGA scaffold	1.0 × 106 human ADSCs	8 wk	Approximately 75%	Shin et al[80]
-Sprague Dawley rats; -Calvarial defect; -5 mm wide	Collagen-resveratrol scaffold	0.05 × 106 human ADSCs	2 wk	Undifferentiated ADSC-seeded construct exhibited better osteogenesis compared to controls and osteoinduced ADSC seeded scaffold	Wang et al[81]
-Athymic nu/nu mice; -Subcutaneous implantation	Alginate microspheres	0.5 × 106 rabbit ADSC	12 wk	Approximately 41%	Man et al[82]
Group 6: Manipulation of recipient host and ADSCs
-Sprague-Dawley rats; -Calvarial defect; -7 mm wide	Polylactic glycolic acid scaffold	1 × 106 human ADSCs	12 wk	Approximately 60%	Wang et al[83]
-C57 black/DBA mice; -Supracondylar right femur defect -0.9 mm wide	Hydrogel	0.3 × 106 mice ADSC	8 wk	Approximately 50%	Deng et al[84]
-Osteoporotic Sprague-Dawley female rats; -Distal epiphysis left femur defect; -3 mm wide	Gelatin	2 × 106 rat ADSCs	5 wk	Approximately 23%	Li et al[85]
Group 7: Allogeneic ADSCs
-New Zealand white rabbits; -Ulna defect; -15 mm	Demineralized bone matrix	60 × 106 rabbit ADSCs	12 wk	Both allogeneic and autologous ADSC seeded construct induced almost complete defect repair while cell-free control remained unrepaired	Gu et al[86]
-Sprague Dawley rats; -Ulna defect; -8 mm long	Demineralized bone matrix	60 × 106 rat ADSCs	24 wk	Radiographs and histology confirmed superior bone healing in the experimental group compared to cell-free control	Wen et al[87]
-Beagle Dogs; -Parietal bone defect; -20 × 20 mm	Coral scaffold	60 × 106 of canine ADSC	24 wk	Approximately 70%	Liu et al[88]
-Wistar rats; -Left radius defect; -4 mm long	Heterogeneous deproteinized bone	0.1 × 106 rat ADSCs	8 wk	Radiographs and histology confirmed improved healing in osteoinduced ADSC/scaffold group compared to undifferentiated ADSC, cell-free, and blank controls	Liu et al[89]
Group 8: Non-manipulated or unaltered ADSCs
Decellularized matrices
-CD1 nude mice; -Distal femur defect -3 mm	Human cancellous bone scaffold	0.5 × 106 human ADSCs	8 wk	hADSCs-seeded scaffold induced significantly superior defect healing compared to cell-free scaffold	Wagner et al[90]
-C57BL/6 mice; -Calvarial defect; -4 mm wide	Extracellular matrix deposited on porcine small intestinal submucosa	0.0025 × 106 of human ADSCs	4 wk	21.77 ± 6.99	Zhang et al[91]
-Institute of Cancer Research mice; -Full-thickness parietal defect; -4 mm wide	Decellularized tendon	1.0 × 106 human ADSCs	8 wk	86%	Ko et al[92]
-Sprague Dawley rats; -Two-wall periodontal intrabony defect; -2.6 × 2.0 × 2.0 mm	Amniotic membrane	0.3 × 106 human ADSCs	3 wk	ADSC-seeded scaffold resulted in a significantly smaller defect size than the control	Wu et al[93]
Ceramics
-Sheep; -Tibia; -3.2 cm long defect	Hydroxyapatite-based particle in a semi-solid milieu	56 × 106 human ADSCs	12 wk	The experimental group showed bridging and significantly better healing compared to control	Ben-David et al[94]
-New Zealand White rabbits; -Full-thickness proximal medial tibia defect; -8 mm wide	Hydroxyapatite	0.2 × 106 rabbit ADSCs	8 wk	The new bone area was equivalent between seeded and unseeded scaffold; however, ADSC seeded construct represented preferable histological characteristics	Arrigoni et al[95]
-New Zealand White rabbits; -Full-thickness proximal medial tibia; -8 mm in diameter	Hydroxyapatite	1.5 × 106 rabbit ADSCs	8 wk	ADSC-seeded scaffold exhibited better scaffold resorption than cell-free scaffold and superior histological characteristics compared to all controls	De Girolamo et al[96]
-Fisher 344 rats; -Calvarial defect; -5 mm wide	Hydroxyapatite	0.4 × 105 rat ADSCs	8 wk	16.88 ± 1.52	Xia et al[97]
-T and B cell-deficient NOD SCID mice; -Subcutaneous implantation	Type I collagen (30%) and magnesium-enriched hydroxyapatite	1 × 106 human ADSCs	8 wk	hADSC-seeded presented improved osteogenesis and angiogenesis compared to cell-free scaffold control	Calabrese et al[98]
-Miniature Pigs; -Mandibular defect -3 cm × 1 cm × 2 cm	Tri-calcium phosphate- poly (D,L-lactide-co-glycolide) scaffolds	5 × 106 porcine ADSCs	12 wk	34.8 ± 4.80	Probst et al[99]
Bioactive glass
-Wistar rats; -Full-thickness calvarial defect; -8 mm wide	Bioactive glass	0.5 × 106 rat ADSCs	12 wk	ADSC-seeded scaffold group exhibited significantly more bone repair and higher bone density compared to blank control. ADSC construct’s result was equivalent to that of autologous bone graft	Saçak et al[100]
-Sprague Dawley rats; -Parietal bone defect; -8 mm wide	Icariin doped bioactive glass	0.5 × 106 rat ADSCs	12 wk	The experimental group saw the complete repair of the defect while all controls showed various degrees of incomplete healing; repair in the experimental group is characterized by mature bone and complete scaffold resorption	Jing et al[101]
Polymers
-Wistar rats; -Calvarial defect; -5 mm wide	Polycaprolactone scaffold	0.05 × 106 human ADSCs	8 wk	Both undifferentiated and osteo-induced ADSC-seeded scaffold resulted in preferable histological features and higher expression of osteogenesis and angiogenesis markers	Caetano et al[102]
Platelet-rich plasma as carrier material
-Beagle dogs; -Tibial defects; -10 mm wide	Activated platelet-rich plasma	1.0 × 106 human ADSCs	6 wk	68.97 ± 0.91	Cruz et al[103]
-F344 rat; -Calvarial defect; -5 mm wide	Activated platelet-rich plasma	0.2 × 106 rat ADSCs	8 wk	95.60	Tajima et al[104]
Hybrid materials
-New Zealand white rabbits; -Calvarial defect; -10 mm wide	Hyaluronic acid-g-chitosan-g-poly (N-isopropylacrylamide) embedded with biphasic calcium phosphate microparticles and PRP	0.1 × 106 rabbit ADSCs	16 wk	The experimental group induced obvious significant bone formation and defect bridging. Cell-free scaffold control showed negligible defect repair	Liao et al[105]
-Sprague Dawley rats; -Parietal defect; -5 mm wide	Multi-layered stacking of electrospun polycaprolactone/gelatin membranes	0.006 × 106 rat ADSCs	12 wk	Up to 90%	Wan et al[106]
-Balb/c nude mice; -Calvarial defect; -4 mm wide	1H,1H,2H,2H-per- fluorodecyl acrylate (97%) and glycidyl methacrylate coated paper scaffold	1.0 × 106 cells/paper human ADSCs	8 wk	92%	Park et al[107]

ADSCs: Adipose-derived stem cells; SVF: Stromal Vascular Fraction; PRP: Platelet-rich plasma.

Table 3 Specific markers used for selection of sub-populations of adipose derived stem cells showing superior bone forming ability

Ref.	Marker	Study outcome and salient findings
	CD146
James et al[110]	CD146+CD34-CD45- (Pericytes) + CD146-CD34+CD45- (Adventitial cells)	Intramuscular ectopic bone formation in SCID mice; FACS purified, human, pericytes + adventitial cells produced significantly more ectopic bone formation than SVF; BMP2 enhanced osteogenic as well as adipogenic differentiation, whereas Nel-1 promoted only bone formation when tested in ectopic bone formation assay; 250000 cells were implanted intramuscularly in SCID mice for 4 wk using collagen sponge or DBX+ β-TCP + 3.5 -11.25 µg of BMP2 or 350 µg Nel-1
James et al[109]	CD146+CD34-CD45- (Pericytes) + CD146-CD34+CD45- (Adventitial cells)	Human pericytes + adventitial cells together make up around 40% of SVF from human lipoaspirate (60 patients tested) both types representing around 20% and these numbers do not change with age, gender, or body mass index; FACS purified, human, pericytes + adventitial cells induce significantly more healing in mouse calvarial defect than SVF; 250000 cells were implanted to critical size (3 mm) calvarial defect in SCID mice for 8 wk using PLGA
Meyers et al[112]	CD146+CD34-CD45- (Pericytes) + CD146-CD34+CD45- (Adventitial cells)	It was feasible to purify human pericytes + adventitial cells using a multi-column approach of magnetic beads; Purified pericytes + adventitial cells could enhance critical size (4 mm) calvarial defect created in SCID mice; 250000 cells were implanted to critical size (4 mm) calvarial defect in SCID mice for 8 wk using PLGA
	CD90
Chung et al[115]	CD90+	CD90+ cells induced almost complete healing of critical size (4 mm) calvarial defect in nude mice compared to CD105low (approximately 75%), CD105high - (approximately 65%), and CD90- (40%) by micro-CT; Taken together CD90+ cells are more osteogenic compared to CD105low cells; 150000 cells were implanted to critical size (4 mm) calvarial defect in SCID mice for 8 wk using PLGA
Ferraro et al[113]	CD90+CD34+	Implantation of human CD90+CD34+ ADSCs in nude mice resulted in the formation of only fat tissue surrounded by loose connective tissue; 250000 cells were implanted subcutaneously in nude mice for 4 wk using a collagen sponge
	CD105
Levi et al[120]	CD105low	FACS-sorted, human, CD105low sub-population of ADSCs significantly enhanced bone regeneration (> 95%) in critical size (4 mm) calvarial defect in CD1-nude mice compared to CD105high (approximately 40%) and unsorted ADSCs (50%-60%); Knockdown of CD105 in ADSCs (shCD105) resulted in improving their ability to induce bone formation (> 60%) compared to ADSCs transfected with control shRNA (30%); 150000 cells were implanted to critical size (4 mm) calvarial defect in nude mice for 8 wk using PLGA-HA
Madhu et al[123]	CD105+CD34-; CD105+ CD34+; CD105-CD34+; CD105-CD34-	FACS-purified, mouse, CD105+CD34− ADSCs that responded maximally to BMPs in vitro failed to induce ectopic bone formation upon their sub‐cutaneous implantation immunocompetent syngeneic mice; FACS-purified CD105-CD34- ADSCs responded the least to BMPs in vitro. A bone marrow-derived, clonal, osteoprogenitor population showing the similar phenotype of CD105-CD34- induced robust bone formation; OM preconditioned 1 × 106 cells were implanted subcutaneously in Balb/c mice for 4 wk using Matrigel
Chan et al[128]	AlphaV+CD200+CD105-D90-	Mouse skeletal stem cells that give rise to bone were identified as AlphaV+CD200+CD105-D90- cells and were present in the femoral growth plate; They were not present in adipose tissue; however, when a collagen sponge loaded with BMP-2 was implanted in adipose tissue, the authors reported de novo formation of AlphaV+CD200+CD105-D90- cells in the adipose tissue; Subcutaneous implantation of 10 µg BMP2+ Collagen Sponge in nude mice for 4 wk
Chan et al[131]	PDPN+CD164+CD73+ CD146-	The human counterpart of mSSC was discovered and was found to be of phenotype PDPN+ CD164+CD73+ CD146-; Human adipose stroma did not naturally contain these cells but when it was mixed with BMP-2 and injected sub-cutaneously it led to skeletal reprogramming and induced formation of PDPN+ CD164+CD73+ CD146- human skeletal stem cells; 10 × 106 cells with 10 µg BMP2 + Matrigel were subcutaneously implanted in nude mice for 4 wk
	CXCR4
Xu et al[133]	CXCR4+	CD146+CD34-CD45- cells were FACS-purified from hard (human periosteum) and soft (adipose and dermal tissue). Cells isolated from hard tissue but not the soft tissues showed a strikingly high tendency for skeletogenesis; This corresponded to high CXCR4 signaling in periosteal cells; Inhibition of CXCR4 signaling abrogated bone-forming potential of CD146+CD34-CD45- periosteal cells; CXCR4+ cells from soft tissue (adipose) derived CD146+CD34-CD45- cells represented osteoblastic/non-adipocytic precursor cells; 1 × 106 cells were implanted intramuscularly in nude mice for 4 wk using DBM putty
	PDGFRα
Wang et al[134]	PDGFRα+	Lineage tracing using PDGFRα reporter mice showed that PDGFRα expression marks different sub-populations in the adipose tissue; PDGFRα+ and PDGFRα− fractions both are multipotent progenitor cells, however, PDGFRα+ ADSCs-derived ectopic implants ossify to a greater degree than PDGFRα− cell fractions; 1 × 106 PDGFRα+ or PDGFRα- cells were implanted intramuscularly in nude mice for 8 wk using HA-β-TCP; Or Subcutaneous implantation of 2.5 µg BMP2 + Matrigel into the inguinal fat pad of PDGFRα+ -CreER for 8 wk

ADSC: Adipose-derived stem cells; FACS: Fluorescence-activated cell sorting; SCID: Severe combined immunodeficiency; BMP: Bone morphogenetic protein; TCP: Tricalcium phosphate; PLGA: Polylactic glycolic acid; HA: Hydroxyapatite; DBM: Demineralized bone matrix.