3.1. Characterization of MSCs

Surface markers are currently being used to identify MSCs for quality control assurance prior to cell preparation, based on ‘good manufacturing practice,’ which is required for investor-mediated clinical developments. Hence, the characterization of MSCs based on surface marker analysis is an essential criterion for the clinical application of BTE methodologies. According to the International Society of Cell Therapy (ISCT) criteria, MSCs express a cluster of differentiation (CD) surface markers such as CD90, CD105, and CD73, but do not express CD11b, CD14, CD19, CD34, CD45, or human leukocyte antigen (HLA)-DR ^[22][23][24][32,33,34].

3.2. Clinical Translation of MSC-Based Bone Regeneration

The basic concept behind a scaffold is to mimic the structure and function of the extracellular matrix (ECM) in tissues. The ECM provides both structural and mechanical stability and regulates some of the core cellular functions ^[25][26][27][42,43,44]. The basic role of scaffolds in BTE is to mimic the ECM of the native bone tissue and provide a functional three-dimensional space for the adhesion, migration, proliferation, and differentiation of osteoblast progenitors in which bone growth can occur ^{[28][29][30][31]}[38,45,46,47]. An ideal scaffold for BTE should substitute for both the structure and function of the ECM and thus be capable of regenerating the lost bone tissue when seeded in conjunction with osteoblast progenitors. BTE innovations have led to the development of new biomaterials that resemble the 3D bone structure, in terms of mechanical properties as well as osteoconductive, osteoinductive, and osteogenic features ^[32][33][48,49]. Traditional bone repair approaches mainly focus on the use of bone grafts from autologous, allogeneic, and xenogeneic sources; however, complications such as donor-site morbidity and host immune rejection limit the application of these tissues ^[34][50]. The promise of BTE has principally involved overcoming these problems. The aims of BTE are to regenerate and restore the function of lost bone tissue using combinations of osteoblast progenitors and synthetic biomaterial scaffolds. Over the past decade, the use of synthetic biomaterials to enhance bone regeneration has significantly developed because of their capacity to mimic the natural environment of the extracellular matrix. The synthetic scaffold biomaterials predominantly used in BTE include calcium phosphate ceramics, biodegradable polymers, and composites, and the combination of ceramics and polymer scaffolds aims to utilize the properties of both materials ^[31][34][35][47,50,51].

3.2.2. Preclinical Studies of BTE in a Large Animal Model Using MSC/Scaffold Combinations

To translate the clinical use of MSCs combined with scaffolds for BTE, large animal model systems that closely resemble human physiology are required. A number of preclinical studies conducted using MSCs with varying combinations of biomaterials in critical bone defect models are listed in Table 1.

Author	Experiment Animal	Type and Size of Defect	Experimental Transplant Groups	Post-Transplant Follow up Period	Outcome

NCT Number	Brief Title	Phase	Conditions	Interventions
Probst et al., 2020 [34]	Mini pigs	Critical mandibular defect (3 × 1 × 2 cm)	3D TCP-PLGA scaffold seeded with osteogenic differentiated Porcine ADSCs (pADSCs).	12 weeks	pADSCs seeded TCP-PLGA scaffold constructs significantly improved bone regenerations compared to empty scaffold.
NCT04297813	Efficacy in Alveolar Bone Regeneration With Autologous MSCs and Biomaterial in Comparison to Autologous Bone Grafting	Phase I	• Alveolar Bone Atrophy	Autologous MSCs and a biomaterial, biphasic Calcium Phosphate (BCP).	Wang et al., 2019 [36]	Rhesus Monkeys	Critical alveolar bone defect (10 × 10 × 5 mm)	3D-Bioactive glass (BG) + BMP/chitosan (CS) + BMMSCs	12 weeks	BMP/CS nanoparticles loaded on 3D-BG scaffold promoted bone regeneration ability in vivo, and preload of BMMSCs promote this ability further.
Humerus, Tibia and Femur Critical sized defects	Combination of HA granules (Bongros®-HA, Bioalpha, Seungnam, Korea), BMP2 and BMMSCs mixed with Plasma solution.	Autologous Bone marrow harvested from posterior Iliac crestal bone	None	Dramatic improvement of bone regeneration compared to preoperative radiographs.
NCT03325504	A Comparative Study of 2 Doses of BM Autologous H- MSC+Biomaterial vs. Iliac Crest AutoGraft for Bone Healing in Non-Union	Phase III	• Non Union Fracture	Culture-expanded autologous BMMSC combined with biphasic calcium phosphate (BCP) biomaterial granules	Hsieh et al., 2019 [37]	Gjerde et al., 2018 [54domestic Ds-Red pigs	Calvarias defect (8 mm in diameter and 2 mm in depth)	Hemostatic gelatin sponge scaffold seeded with EGFP pig BMMSCs	]	Severe mandibular ridge resorption.	Expanded, autologous MSCs with biphasic calcium phosphate (MBCP+
NCT02803177	1, 2, 3 and 4 weeks	Osteoid formation in the scaffolds transplanted with seeded BMMSCs was significantly higher than the control group.
Cell Therapy by Autologous BMC for Large Bone Defect Repair	TM; Biomatlante, France)	Bone marrow cells from the posterior iliac crest	Shi et al., 2019 [38]	Minipigs	Maxillary Intraosseous circular defects (12 mm in diameter and 5 mm in depth)	Bio-Oss/autogenous (Pig Gingival MSCs) pGMSCs (2 × 106)/SB431542 (TGF-β signalling inhibitor).	8 weeks	pGMSCs treated with a TGF- β signaling inhibitor successfully repair minipig severe maxillofacial bone defects.
None	MSCs successfully induce significant new bone formation	Qiu et al., 2018 [39]	Minipigs	Lateral femoral condyle defect (8 mm in diameter and 10 mm in depth)	Calcium phosphate cement (CPC) scaffold seeded with autologous BMMSCs plus autologous PRP (CPC-BMSC-PRP, 1 × 106 cells/scaffold)	6 and 12 weeks	CPC scaffold co-delivered BMMSCs-PRP promoted scaffold resorption and doubled bone regeneration in large defects than control groups
Zhang et al., 2017 [40]	Minipigs	Non-healing full thickness cranial defects (2 cm width × 3 cm length × 0.5 cm depth)	IMC (intrafibrillarly-mineralized collagen) scaffold seeded with 1 × 106 PDLSCs cells	12 weeks	Compared with HA, IMC-seeded PDLSCs achieved a significantly higher extent of new bone formation, with the normal architecture of natural bones and blood vessels.
Scarano et al., 2017 [41]	Minipigs	Critical-size circular defects (5 mm diameter; 5 mm thickness) in the mandibular body	Bone porcine block (BPB) scaffold seeded with 100 ul cell suspension of BMMSCs	12 weeks	BPB when used as a scaffold induce bone regeneration and further benefit from the addition of BMMSCs in the tissue-engineered constructs.
Lin et al., 2015 [42]	Minipigs	Massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora	Transduced pig ADSCs loaded onto PLGA scaffold	2, 4, 8 and 12 weeks	ADSCs/scaffold constructs successfully healed massive segmental bone defects at the mid-diaphysis of femora in minipigs significantly than control group.
Cao et al., 2015 [43]	Mini pigs	Calvarial bone defects (3 cm × 1.8 cm oval defect)	BMMSCs pretreated with 75 μg/mL aspirin for 24 h seeded onto hydroxyapatite/tricalcium phosphate (HA/TCP)	6 months	BMMSCs pretreated with aspirin have a greater capacity to repair calvarial bone defects in a mini swine model
Fan et al., 2014 [44]	Rhesus monkeys	Segmental tibial defects (20 mm in length)	Autologous prevascularized BMMSCs (5 × 106)-β-TCP constructs	4, 8 and 12 weeks	Significantly higher amount of neo-vascularization and radiographic grading score in prevascularized BMMSCs-β-TCP constructs

3.2.3. Gene Therapy for Bone Regeneration

Gene therapy is another promising approach for enhancing bone regeneration. Today, the advancement in life-sciences technology allows gene transfer technology to fabricate a tissue-engineered scaffold to accommodate the growth of genetically modified cells and the endogenous synthesis of desired gene products in a controlled manner. Gene therapy allows for the transfer of genetic material in the precise anatomic location of target cells, allowing the transgene expression from the cells with the currently available techniques ^[45][76]. Gene transfer can be performed by several ex vivo and in vivo delivery techniques and by either using viral (transduction) or non-viral (transfection) vectors ^[45][46][76,77]. Since this review is focused on the use of combined cell scaffold constructs for bone regeneration, we mainly discuss the ex vivo delivery method, which requires isolation of target cells and transfer of the desired gene to express the respective protein in vitro and then seeded onto the biocompatible carrier material to obtain cell-scaffold construct for bone tissue engineering applications. The two standard methods of ex vivo delivery include viral and non-viral, it being said that each type has its advantages and disadvantages. Viral vectors demonstrate high transfection efficiency with immunogenicity and toxicity, raising an issue of safety. In contrast, non-viral vectors usually consist of plasmid or related DNA, which are non-immunogenic and high safety but with low transfection efficiency ^[46][47][77,78]. Another promising approach is the sequential delivery of exogenous genes to promote the osteogenesis of stem cells. For example, genes that are expressed early and in the final stages of osteogenesis are different. Hence, delivering required osteogenic genes at specific time intervals into target cells induces efficient osteogenic differentiation. A recent study by Kim et al. demonstrated an effective sequential delivery of runt-related transcription factor 2 (RUNX2) and osterix genes induced conversion of human MSCs into pre-osteoblasts and subsequent delivery of activating transcription factor 4 (ATF4) gene triggered further osteogenesis. Differentiation of MSCs into desired mature cells can be regulated by the delivery time of specific osteogenic genes mimicking the natural process of bone remodeling ^[47][48][78,79].

3.2.4. Clinical Trials of MSCs for BTE

Over the past decade, a greater understanding has emerged with regard to the capabilities of MSCs to promote bone tissue regeneration, with numerous preclinical and clinical studies now underway. To identify the current potential combination of cell-scaffold constructs or tissue-engineered substitutes for bone tissue regeneration, we found twenty clinical trials. Nine are published (Table 2), and others are listed in the ClinicalTrails.gov database (Table 3). These trials have highlighted the importance of using cell-based therapy with various scaffolds to treat bone tissue regeneration in a real clinical setting. From the twenty identified clinical studies listed in Table 2 and Table 3, the majority report the use of BMMSCs, reflecting the fact that they are the most accepted cell source and the current gold standard in most clinical trials for treating bone disease, including nonunion fractures of long bones and craniofacial bone defects. However, in a few clinical trials, researchers have used umbilical cord (UC)- MSCs ^[49][85], BMMSCs ^[50][86], and adipose-derived MSCs as allogeneic cell sources to prepare the tissue-engineered constructs for regeneration of critical bone defects (NCT02307). Ceramic-based scaffolds are the primary choice in the majority of clinical trials, indicating their high clinical relevance. From the clinical trials listed in Table 2 and Table 3, most studies used a combination of BMMSCs with calcium-phosphate ceramics such as hydroxyapatite ^[49][51][52][85,87,88], β-TCP ^[50][53][86,89] (NCT02803177, NCT02153372), biphasic calcium phosphate, a combination of hydroxyapatite and β-TCP ^[54][90] (NCT04297813, NCT03325504, NCT01842477). Although most of these clinical trials used a simple combination of calcium-phosphate ceramics with BMMSCs, in a few studies, however, additional factors were included to facilitate enhanced bone regeneration. For example, Dilogo et al. added growth factor BMP2 along with cell scaffold constructs to enhance bone regeneration ^[49][52][85,88]. Similarly, researchers used BMMSCs mixed with BMP2 and loaded them on to 3-dimensional tissue-engineered collagen scaffold (NCT01958502) in another clinical trial. However, a clinical study by Baba et al. used polylactic scaffold seeded BMMSCs mixed with platelet-rich plasma solution and an additional 5000 units of human thrombin dissolved in 10% calcium chloride ^[55][91].

Author	Type and Size of Defect	Transplant Groups	Origin of Cell Source	Pre-Transplant Incubation	Outcome
Dilogo et al., 2020 [49]	Nonunion fractures of Humerus/tibia with critical size bone defects	Combination of HA Bongros®-HA, Daewoong), BMP2, UC-MSCs with demineralized bone matrix	Allogeneic Umbilical Cord MSCs (UC-MSCs)	None	Allogeneic UC-MSCs can be used safely to treat the critical sized bone defects of long bones.
Dilogo et al., 2019 [52]
Phase II	• Humerus Fracture Displaced Proximal	Baba et al., 2016 [55]	Intrabony Periodontal defect. Probing depth >4 mm	The mixture of BMMSCs and PRP, combined with human thrombin dissolved in 10% calcium chloride perfused in a 3D woven-fabric composed of poly-L-lactic acid resin fibers (MSCs/PRP-3D woven Fabric)	Autologous Bone marrow harvested from posterior Iliac crestal bone	Induced under Osteogenic Medium	BMMSCs/PRP-3D woven Fabric constructs showed efficient regeneration of the periodontal tissue including alveolar bone.
Morrison et al., 2018 [50]	Cranial defects with less than 80 mm diameter	Allogeneic mesenchymal stromal cells (MSCs) on a ceramic carrier (ChronOS granules, synthes, and polymer scaffold,	Allogenic BMMSCs from 18–25 years aged donors	None	Allogeneic MSCs can be safely used for bone regeneration.
Autologous Bone Marrow-derived Mononuclear Cells (BMC) seeded onto ß-TCP	Kaigler et al., 2015 [53]	Severe Bone Atrophy of upper Jaw	Combination of BMMSCs and β-TCP (Cerasorb, Curasan AG, Germany)	Autologous Bone marrow harvested from posterior Iliac crestal bone	None	Higher density of regenerated bone with MSCs+ β-TCP group was observed than control group.
NCT02307435	Marcacci et al., 2007 [51]	Humerus, Tibia and ulnar Critical sized defects	Combination of invitro expanded BMMSCs seeded with porous hydroxy apatite scaffolds (Finblock, FinCeramica Srl, Faenza, Italy)	Autologous Bone marrow harvested from posterior Iliac crestal bone	None	Significant healing of the CSDs. Attained long term durability of bone regeneration.
Allogenic Mesenchymal Stem Cell for Bone Defect or Non Union Fracture	Early Phase I	• Non Union Fracture, Metaphyseal Fibrous Defect	Allogeneic MSCs from umbilical cord/bone marrow/adipose combined and HA-CaSo4
NCT02153372	Cell Therapy by Bone Marrow- derived Mononuclear Cells (BMC) for Large Bone Defect Repair: Phase-I Clinical Trial	Phase I	• Humerus Fracture Displaced Proximal	Autologous Bone Marrow-derived Mononuclear Cells (BMC) seeded onto ß-TCP
NCT01958502	Evaluation the Treatment of Nonunion of Long Bone Fracture of Lower Extremities (Femur and Tibia) Using Mononuclear Stem Cells from the Iliac Wing Within a 3-D Tissue Engineered Scaffold	Phase II	• Nonunion of Fracture	BMMSCs with BMP2 within a 3-D tissue engineered collagen scaffold
NCT01842477	Evaluation of Efficacy and Safety of Autologous MSCs Combined to Biomaterials to Enhance Bone Healing	Phase I/II	• Delayed Union After Fracture of Humerus, Tibial or Femur	BMMScs mixed with biphasic calciulm granules	Bajada et al., 2007 [56]	Tibial non-union	Combination of invitro expanded BMMSCs seeded with calcium sulphate pellets (Stimulan, Biocomposites Ltd., Keele, United Kingdom)	Autologous Bone marrow harvested from posterior Iliac crestal bone	None	Clinical and radiological healing of nonunion was observed
NCT00250302	Autologous Implantation of Mesenchymal Stem Cells for the Treatment of Distal Tibial Fractures	Phase I/II	• Tibial Fracture	BMMSCs loaded onto a carrier and implanted locally at the defect site	Morishita et al., 2006 [57]	Tibial/femur massive defects	Attachment of invitro expanded BMMSCs-HA granules	Autologous Bone marrow harvested from posterior Iliac crestal bone	Induced under Osteogenic Medium
NCT00557635	Good integration of BMMSCs-HA constructs to the host bone and increased radiographic density of the defect area.

Osseous Setting Improvement With Co-implantation of Osseous Matrix and Mesenchymal Progenitors Cells From Autologous Bone Marrow
Phase II
• Tibia or Femur Pseudo-arthrosis
Injection of an osseous matrix (osteopure) combined with MSC progenitors from autologous bone marrow.
NCT02177565	Autologous Stem Cell Therapy for Fracture Non-union Healing	Not available	• Non-union of Fractures	Autologous BMSCs combined with carrier material
NCT01435434	Mononucleotide Autologous Stem Cells and Demineralized Bone Matrix in the Treatment of Non Union/Delayed Fractures	Not available	• Non Union/Delayed Fractures	Injection of Autologous Stem Cells and Demineralized Bone Matrix

4. Osteoblast-Based Bone Tissue Regeneration

In addition to the efforts to increase the bone-forming ability of MSCs as a cell source for bone tissue engineering, the use of osteoblasts that are capable of proliferating before maturing, and that can synthesize and deposit bone extracellular matrix components such as osteocalcin (OCN) and bone sialoprotein (BSP), provides a potential alternative BTE cell source for the treatment of large bone defects. However, since BTE is generally approached using a combination of osteoblasts induced from MSCs on biodegradable scaffolds, the resulting bone forming efficacy will be dependent on the differentiation potential of MSCs into osteoblasts. This could hamper the progress of BTE for treating large bone defects. There are two major mechanisms underlying skeletal development, intramembranous and endochondral ossification. In intramembranous ossification, osteoblast lineage cells, i.e., immature osteoblasts, are formed directly from condensed mesenchymal tissue. Endochondral ossification, by contrast, involves the production of osteochondral progenitors from MSCs that give rise to hyperchondrocytes which activate perichondrial cells to differentiate into immature osteoblasts. From the perspective of BTE, the formation of immature osteoblasts is the convergence point for both types of ossification.

5. Conclusions

Critical bone defects that cannot self-heal without a surgical intervention pose a significant challenge in the field of BTE. Compared to the traditional gold standard approach of using autogenous bone, regenerative methods will typically use either exogenous MSCs or immature osteoblasts seeded onto a bioactive scaffold placed at the defect area to regenerate functional bone. Adult MSCs from bone marrow and adipose tissue have often been used in various clinical studies for bone regeneration. The available data from both preclinical studies and clinical trials have shown promising results when BMMSCs are used as a cell source for bone tissue regeneration. Many clinical studies have also shown the beneficial effects of MSCs and scaffold combinations in bone healing. Although encouraging clinical results have been obtained by transplanting MSCs-scaffold constructs, the exact dosage and route of application remain to be optimized, and the fate of transplanted cells and their mechanisms of action need to be better monitored in more extensive future clinical trials. The development of an alternative immature osteoblast source combined with more effective scaffolds is also anticipated in the future. Immature osteoblasts have the ability to become a potential alternative cell source to adult MSCs, as they are osteogenic lineage-committed cells that enhance the efficacy of bone regeneration.

Immature osteoblasts can be obtained from bone tissue samples during routine oral surgical procedures from mandible/maxillary alveolar bone or surgeries involving long bones from the femur or tibia. The advantage of immature osteoblasts over MSCs is their spontaneous matrix formation upon transplantation without promoting osteogenic differentiation. Immature osteoblasts can directly secrete bone collagen matrix and release various factors such as M-CSF, RANKL, and VEGF, enhancing bone remodeling and bone regeneration in various BTE applications. The combination of an immature osteoblast culture system that possesses robust osteogenic activity and an appropriate biodegradable scaffold is an expected future BTE therapeutic option. This approach will facilitate the establishment of better clinical protocols for regeneration therapy in cases of large bone defects, as a treatment for orthopedic conditions such as back pain resulting from stenosis and lumbar spondylolisthesis osteosarcoma, and for horizontal alveolar bone defects in the dental field.