Table 1.

Current progress in the development of osteochondral tissue-engineered scaffolds using conventional manufacturing approaches

Scaffold categories		Composition	Brief fabrication strategy	Key results	Refs
Stratified	Bi-phasic	Bone layer: HAp Cartilage layer: HA hydrogel KGN and ALN loaded in cartilage and bone layers	Semi immersion technique. ALN-loaded HAp scaffolds were kept in a mold partially immersed in gelatin. In refrigerated conditions, the KGN-loaded pre-crosslinked HA hydrogel was poured on the top to infiltrate the leftover height of the HAp scaffold and fully UV crosslinked. Later the gelatin was removed from the bottom zone of the HAp scaffold in warm water.	1. Micro-computed tomography: efficient integration of the layers. hBMSc were cultured in the scaffolds	[101]
				2. In the KGN-HA phase, the expression of aggrecan, collagen II upregulated after 21 days of culture. Expression levels of ALP, RunX2 and collagen I in the ALN-HAp phase were also upregulated.
				3. rMSCs were seeded in the scaffolds and implanted subcutaneously for 2 months. Expression levels of all the chondrogenic and osteogenic marker genes were significantly higher compared to the drug-free scaffolds.
		Bone layer: bovine bone-derived ECM hydrogel Cartilage layer: bovine cartilage-derived ECM hydrogel	The individual ECMs were stacked on top of each other in a cylindrical mold. The ECM hydrogel complex was lyophilized together to obtain the bi-phasic osteochondral scaffold. Bone marrow-derived stem cells (BMSCs) were seeded in the bi-phasic scaffolds.	1. A significantly higher expression of the chondrogenic (aggrecan, collagen II and SOX9) and osteogenic marker genes (collagen I, OCN, RUNX2 and ALP) in the case of the ECM-based scaffolds compared to the untreated control groups	[102]
				2. In vivo histological staining (H&E, Toluidine blue, Safranin-O and fast green) and immunohistochemistry (collagen I and collagen II) showed that the ECM-based scaffolds surpassed the untreated controls
				3. ICRS and O’Driscoll scores were consistently higher in the bi-phasic ECM-based scaffold compared to the untreated control group
		Bone layer: SF and CaP composite Cartilage layer: SF	Salt leaching and freeze-drying. CaP nanoparticles by acid-base reaction in silk solution in presence of NaCl particles. NaCl particles are leached and fresh SF solution is added on the top of the microporous SF-CaP scaffold.	1. Enhanced attachment, viability and proliferation of RBMSCs in vitro	[107]
				2. Host tissue integration in rabbit critical OC defect with a layer of connective tissue adhered on the entire surface of the scaffolds, no signs of acute inflammation
				3. The biphasic scaffolds showed cartilage regeneration in the silk layer, along with subchondral bone ingrowth and angiogenesis
		Bone layer: PCL Cartilage layer: methacrylated HA	Salt leaching, infiltration and photocrosslinking. PCL was dissolved in chloroform with NaCl particles. The solvent evaporated, NaCl leached and the porous PCL scaffold was suspended in LAP-modified MeHA solution with bovine MSCs. Finally, the gel is UV photo-crosslinked	1. The mechanical properties of the MSCs encapsulated HA hydrogel/PCL osteochondral scaffolds matured over time, and obtained the peak compressive strength at a time when hydrogel/PCL interfacial shear strength was still developing.	[117]
				2. In a TGF-β3 supplemented culture media, the GAG (Safranin O/Fast green and alcian blue) and collagen (picrosirius red) contents increased over time indicating the zone-specific differentiability of MSCs.
	Multi-phasic	Bone layer: PLGA-BG Intermediate layer: agarose and PLGA-BG Cartilage layer: agarose gel	BG-PLGA microspheres were sintered and agarose gel was partially infiltrated to make the transition region while pure agarose gel was retained on the top as the cartilage phase. Chondrocytes were laden in agarose and the PLGA-BG phase was seeded by osteoblasts	1. During co-culture, both chondrocytes and osteoblasts maintained their phenotypes, where chondrocytes produced proteoglycans and type II collagen while the osteoblasts deposited type I collagen and maintained ALP activity.	[110]
				2. Calcified interface generation was observed in the transition zone
				3. Increment in chondrocyte density resulted in elevated ECM deposition and higher mechanical properties
		Bone layer: HAp Intermediate layer: 20% SF Cartilage layer: 5% SF	HAp-5% SF solution casted in mold, lyophilized and sintered for porous HAp. Next, 20% SF solution was added on top of the porous structure followed by freeze drying to obtain the calcified cartilage layer. Finally, 5% SF solution was added on top of it and lyophilized. PDA modification of scaffolds and the cartilage layer was laden with PDGF. SMSCs were seeded in the scaffolds.	1. Capability of osteochondral regeneration was in the trochlear zone of the rabbit knee joint	[111]
				2. The in vitro PDGF release in the PDA-modified scaffolds was sustained in nature
				3. In agreement with MRI and gross morphological assessment, histological staining (H&E, Safranin O/fast green) and immunohistochemistry (col I, col II, aggrecan) analysis demonstrated significantly higher regeneration capability of the PDA-PDGF scaffolds, in vivo (trochlear groove of rabbit femur)
		Bone layer: HAp-SF Intermediate layer: cell-free intermediate layer induced in vitro Cartilage layer: 6% SF	HAp and SF solution infiltrated in partially sintered paraffin microspheres in a mold and frozen. 6% SF solution was poured on top of it and directionally solidified. Next, the entire unit was lyophilized. Columnar SF phase is crystallized with methanol and partially sintered paraffin microspheres were leached out with hexane leaving interconnected microporosities in the bony layer.	1. TIPS induced bionic columnar cartilage zone seamlessly with a simulated bone layer	[112]
				2. A cell-free compact region in between the cartilage and subchondral bone was observed which closely resembles the intermediate calcified cartilage in normal osteochondral tissue
				3. In the chondral region, toluidine blue, Safranin O and immunohistochemical staining revealed the continued GAG deposition and collagen II expressions over time
				4. Alizarin red, von Kossa and immunohistochemical staining in the bony zone revealed a time-dependent increment in matrix mineralization and collagen I expression
Gradient		PLGA-HAp microsphere-based gradient scaffold with HA hydrogel infiltrated in the higher porous distal (cartilage) end	HAp-modified PLGA microspheres with varying vol% of NaCl stacked in a mold followed by sintering. NaCl was leached in water and the higher porosity end (distal) was infiltrated with HA hydrogel. PLGA-HAp scaffolds were pre-modified with BMP-2 and the hydrogel contained TGF-β1 along with hMSCs. Scaffolds were cultured in hMSCs laden co-differentiation media	1. Higher GAG deposition was observed in the distal cartilage end whereas there was no significant difference in the osteogenesis at the two ends of the scaffolds	[68]
				2. Synergistic effect of BMP-2 and TGF-β1 in chondrogenesis was established
				3. Col II, Sox9, GAGs and aggrecan expressions increased with BMP-2 in a dose-dependent manner
		PLGA-based scaffolds having opposing gradients of chondroitin sulfate (CS) and β-TCP	PLGA-CS and PLGA-TCP monodispersed microspheres were separately suspended and injected from mutually opposite directions in a cylindrical mold. The suspension media was filtered out and the gradient chondrogenic and osteogenic scaffold was sintered at ambient temperature using the ‘solvent/non-solvent’ sintering technique (ethanol/acetone).	1. From H&E and Safranin-O staining it was found that the in vivo cartilage regeneration in the scaffolds with TGF-β3, was significantly higher compared to the group who received IGF-1 modified PLGA-CS-TCP scaffolds.	[113]
				2. First long-term study in literature for critical-sized gradient osteochondral scaffolds in a large animal model where ‘microfracture’ was used as the control treatment
				3. Hyaline cartilage regeneration in unmodified and growth factor modified PLGA-CS-TCP scaffolds overwhelmed the microfracture treated groups
		GelMA with density modifier Ficoll as the base layer and HepMA as injected phase for a buoyancy-driven gradient.	A lower density HepMA phase was injected in a higher density GelMA base layer and after the formation of the stable buoyancy-driven gradient solution, the entire system was immobilized using UV crosslinking. The HepMA phase was pre-laden with BMP-2 and both the phases were pre-loaded with hMSCs and photoinitiator.	1. Alizarin red S staining showed the formation of osseous cap formation in the BMP-2 rich phase (HepMA side) while alcian blue revealed the sulfated GAGs throughout the length of the scaffolds	[116]
				2. Similar trend in immunohistochemical analysis, collagen type II expression throughout the scaffold and osteopontin expressed in the mineralized end
				3. Raman spectroscopic analysis manifested the fingerprints of hydroxyapatite (predominant) and β-TCP in the mineralized layer