. 2021 Mar 30;12:20417314211004211. doi: 10.1177/20417314211004211

Table 1.

Cartilaginous constructs engineered by chondrogenic priming alone for bone defect reconstruction.

Reference	Cell source	Biomaterial	In vitro priming condition	Bone defect model	Highlighted results
Iimori et al. (2021)¹⁷²	hiPSC line	/	Scaffoldless suspension culture; 10, 12, or 17 weeks in CHM containing TGF-β₁, BMP-2, and GDF-5	3.5-mm femoral defect in SCID mice	The hiPSC-derived cartilage produced new bone via reminiscent of SOC-ECO process in the defects.
Iimori et al. (2021)¹⁷²	hiPSC line	/		3.5-mm femoral defect in SCID mice	Less time for chondrogenic differentiation of hiPSCs resulting in faster bone formation.
Longoni et al. (2020)⁴⁴	hBMSCs; rat BMSCs	COL I gel	Spheroid culture; 4 weeks in CHM containing TGF-β₁ and BMP-2	6-mm femoral defect in Brown Norway rats	The amount of endochondral bone formation was proportional to the degree of host-donor relatedness.
Longoni et al. (2020)⁴⁴	hBMSCs; rat BMSCs	COL I gel		6-mm femoral defect in Brown Norway rats	No full bridging of the defect was observed in the hBMSCs group, whereas 2/8 and 7/7 bridges formed in allogeneic and syngeneic group, respectively.
Nilsson Hall et al. (2020)⁶⁰	hPDCs	/	Microspheroid culture; 4 weeks in chemically defined CHM containing BMP-2, TGF-β₁, GDF-5, BMP-6, and FGF-2	4-mm tibial defect in NMRI^nu/nu mice	Engineered callus organoids spontaneously bioassembled in vitro into large, engineered tissues able to heal murine critical-sized long bone defects via ECO.
Freeman et al. (2020)³⁶	hBMSCs; hUVECs	PCL scaffold	Endochondral priming: 3 weeks in CHM containing TGF-β₃; Osteogenic priming: 3 weeks in osteogenic medium	4-mm calvarial defect in immunocompromised mice	The addition of hUVECs alone or a coculture of hUVECs and hBMSCs did not benefit for either the vascularization or mineralization potential of the scaffolds.
Freeman et al. (2020)³⁶	hBMSCs; hUVECs	PCL scaffold		4-mm calvarial defect in immunocompromised mice	Endochondral priming alone was sufficient to induce vascularization and subsequent mineralization.
Wang et al. (2018)¹¹⁹	Rat BMSCs	HAp-coated porous Ti₆Al₄V scaffolds	4 weeks in CHM	5-mm full-thickness circular mandibular defect in SD rats	The HAp-coated Ti₆Al₄V scaffolds improved the chondrogenic differentiation of BMSCs in vitro and increased new bone formation via ECO in vivo.
Daly et al. (2018)⁹⁷	Rat BMSCs	GelMA hydrogel with 3D printed microchannels	4 weeks in CHM containing TGF-β₃ and BMP-2	5-mm femoral defect in Fischer rats	3D-printed hypertrophic cartilage grafts with microchannels promoted osteoclast/immune cell invasion, hydrogel degradation, and vascularization following implantation.
Bolander et al. (2017)⁶¹	hPDCs	COL I gel	Cell aggregate culture; 6 days of preconditioning in serum-free CDM or growth medium followed by 6 days of stimulation by BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, and GDF-5 in CDM	4-mm tibial defect in NMRI^nu/nu mice	Serum-free preconditioning in CDM enhanced BMP-2-induced osteochondrogenic differentiation of PDCs.
Bolander et al. (2017)⁶¹	hPDCs	COL I gel		4-mm tibial defect in NMRI^nu/nu mice	Combined in vitro priming by BMP-2 treatment and aggregation led to endochondral bone formation and critical-size bone defect healing in vivo.
Bardsley et al. (2017)⁷⁹	Rat nasal chondrocytes	PGA scaffold	Constructs were cultured in basic medium containing insulin and ascorbic acid for 5 weeks	4-mm full-thickness calvarial defect in Wistar rats	Constructs derived from nasal chondrocytes had the capacity to express features of hypertrophic chondrocytes.
Bardsley et al. (2017)⁷⁹	Rat nasal chondrocytes	PGA scaffold		4-mm full-thickness calvarial defect in Wistar rats	Nasal chondrocytes can be used to engineer hypertrophic cartilage and repair bone defects.
van der Stok et al. (2014)¹⁷³	hBMSCs	/	Pellet culture; undifferentiated pellets: 3 days in CHM containing TGF-β₁; Chondrogenically differentiated pellets: 3 weeks in CHM containing TGF-β₁	6-mm femoral defect in RUN rats	Chondrogenically differentiated pellets resulted in significantly more bone and vascularization in critical bone defects through ECO than undifferentiated pellets.
Harada et al. (2014)¹⁰⁸	Rat BMSCs	PLGA scaffold	3 weeks in CHM containing TGF-β₃ and BMP-2	5-mm or 15-mm femoral defect in Fischer rats	The large 15-mm implants reached 75% of the strength of the normal rat femur, while there was no significant difference in the strength of the 5-mm implants.
Mikael et al. (2014)¹⁷⁴	hBMSCs	Donut-shaped Healos scaffold disc	Pellet culture, 16 days in CHM containing TGF-β₁	3.5-mm calvarial defect in NSG mice	Precartilage template formed in vitro induced mineralized tissue formation via a cartilage-mediated process.
Bahney et al. (2014)⁷⁰	hBMSCs; hACs	PEGDA scaffolds	Pellet culture, 3 weeks in CHM containing TGF-β₁;	2-mm segmental tibial defect in Nude mice	Cartilage grafts from fracture callus produced well-vascularized and integrated bone regeneration via ECO in bone defects.
			Scaffold culture, 6 weeks in CHM containing TGF-β₁		hBMSC-derived cartilage pellets promoted bone regeneration via ECO in bone defects.
			Scaffold culture, 6 weeks in CHM containing TGF-β₁		Both hBMSC and hAC-encapsulated PEGDA scaffolds synthesized COL II and sulfated proteoglycans, but only hBMSC-encapsulated PEGDA scaffolds elaborated COL I and X proteins.
Jukes et al. (2008)⁶²	Mouse ESC line IB10	Ceramic scaffolds	3 weeks in serum-free CHM containing TGF-β₃	8-mm calvarial defect in immunodeficient rats	Significantly more bone ingrowth was observed in the inner circle of the tissue-engineered cartilaginous constructs.
Huang et al. (2006)¹⁷⁵	Rabbit BMSCs	Composite sponge of 70% esterified hyaluronan and 30% gelatin	3 weeks in serum-free CHM containing TGF-β₁	Lunate excision in adult New Zealand white rabbits	Cartilaginous implants formed abundant bone tissue and blood vessels through ECO.

BMSCs, bone marrow-derived mesenchymal stem cells; CDM, chemically defined medium; CHM, chondrogenic medium; ESCs, embryonic stem cells; GDF-5, growth/differentiation factor 5; GelMA, gelatin-methacrylamide; hACs, human articular chondrocytes; HAp; hydroxyapatite; hiPSCs, human induced pluripotent stem cells; hPDCs, human periosteum-derived cells; hUVECs, human umbilical vein endothelial cells; PCL, poly(ε-caprolactone); PEGDA, poly(ethylene glycol) diacrylate; PGA, polyglycolic acid; PLGA, poly(lactic-co-glycolic acid).

CHM is typically defined as DMEM supplemented with 100 U/mL penicillin/streptomycin, 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 50 μg/mL L-ascorbic acid 2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL BSA, 1× ITS, 100 nM dexamethasone, and 10 ng/mL human TGF-β₁ or TGF-β₃.