Indication
Testing condition
Result
Reference
In vitro
In vivo
In vitro
In vivo
To verify the capability of hAFSCs of osteogenic differentiation forming tissue-engineered bone
Printed hAFSC-alginate/collagen scaffold are differentiated to osteogenic lineage in osteogenic medium
Subcutaneous implantation of ASFCs/scaffold into immunodeficient mice
Osteogenic differentiation: mineralization, ALP activity and gene expression RUNX2, OCN, ALP
– Ectopic bone formation (hard mineralized tissue generation after 18 weeks)
De Coppi et al. [17]
To verify the capability of hAFSCs to osteogenic differentiation
Differentiated in osteogenic medium
Differentiation of osteoblast with calcium mineralization (von Kossa stain)
Kim et al. [31]
To see the potential of AFSCs to produce 3D mineralized ECM within porous mPCL scaffold
– hAFSCs initially differentiated in 2D culture in osteogenic medium
Subcutaneous implantation of pre-differentiated AFSCs/mPCL scaffold into athymic rats
– 80 % of colonies are mineralized
– Pre-differentiated AFSCs produce seven times more mineralized matrix
Peister et al. [32]
– 2D-cultured hAFSC seeded in 3D mPCL scaffold
– AFSC are viable after 15 weeks in 3D culture
– AFSCs produce mineralized bioengineered constructs in vitro and in vivo
To repair bone defect (to determine osseous grafts engineered from AFSCs)
Rabbit AFSCs seeded in PLLA electrospun nanofiber in bioreactor
Implantation of pre-differentiated electrospun nanofibrous scaffold to the defective intercostal space
Ectopic bone formation: full thickness repair of sternal defects, graft density increased
Steigman et al. [33]
Osteogenic differentiation of hAFSCs in nanofibrous scaffold induced by rhBMP-7
hASFCs on the NF scaffolds induced by 50 ng/mL rhBMP-7
Subcutaneous implantation into athymic nude mice
– Osteogenic differentiation by mineralization (calcium) and expression of gene (RUNX2, OSX)
Ectopic bone formation
Sun et al. [30]
– Synthetic nanofibrous scaffolds enhance osteoblastic differentiation of hAFSCs
– rhBMP-7 induce mineralize hAFSCs more than MSCs
Compare osteogenic capacity of hMSCs and hAFSCs, on electrospun nanofiber meshes
hMSCs and hAFSCs are seeded in 3D nanofiber mesh in osteogenic medium
hAFSC show delayed ALP peak, but elevated mineral deposition, compared to hMSCs
Kolambkar et al. [18]
To explore the feasibility of fabrication a macroscopic bone tissue at centimeter scale
Two stage culture
Osteogenic differentiation: mineralization (calcium), ALP, expression of gene (collagen type I, OCN)
Chen et al. [34]
– hAFSC-laden CultiSpher S microcarrier (from porcine gelatin) to prepare modular tissue
Good viability and homogenous distribution of AFSCs and bone characteristic ECM
– Assembly of microcarrier in cylindrical perfusion culture chamber
To compare osteogenic differentiation capacity between MSCs and AFSCs
A1hAFSC and H1hAFSC seeded within mPCL scaffold
AFSCs have delayed, robust osteogenic differentiation compared to MSC
Peister et al. [23]
– 2D culture in osteogenic medium and hAFSC seeded in 3D mPCL/collagen gel scaffold
To repair critical size cranial bone defects (to evaluate fibroin scaffolds with hAFSCs and hDPSCs)
Fibroin scaffold seeded with hAFSC or hDPSCs in osteogenic medium
Cell/scaffold implant into critical-sized cranial defect in immunocompromised rat
Osteogenic differentiation by radiology, confocal microscopy, histologic method
Bone formation and defect correction with higher bone formation by hAFSC-seeded scaffold
Riccio et al. [35]
To investigate BSM with 3D PLGA scaffold to facilitate osteogenic differentiation
hAFSC seeded in 3D composite scaffold system using collagen matrix from porcine BSM and PLGA in osteogenic media
Osteogenic differentiation: mineralization, ALP activity and gene expression RUNX2, OPN, OCN
Kim et al. [22]
The majority of studies have both proven that AFS cells are capable of differentiating into osteoblasts in 3D and confirmed that, following induction, these stem cells have potential therapeutic utility. Kim et al. [22] demonstrated that differentiation of human AFS cells were induced in the 2D osteogenic medium, containing dexamethasone, ascorbic acid, and β-glycerophosphate. This was evidenced by both qualitative and quantitative analysis of Alizarin Red staining that indicates calcium deposition as shown in Fig. 10.1. Subsequently, a composite scaffold using a collagen matrix derived from porcine bladder submucosa matrix (BSM) and poly(lactide-co-glycolide) (PLGA) was developed and studies were performed to determine whether human AFS cells seeded in this composite scaffold retained their ability to induce osteogenic differentiation in the osteogenic medium. The results from these studies showed that human AFS cells adhered to the composite scaffolds with uniform distribution and proliferated over time. Osteogenic differentiation on the composite scaffold was also assessed using real-time PCR, and expression levels of three major transcription factors associated with osteogenic differentiation, RUNX2, OPN, and OCN, were consistently higher in the scaffold groups treated with osteogenic medium than untreated scaffolds. Mineralization of the differentiated samples was also confirmed by calcium content measurement.
Fig. 10.1
Effect of osteogenic medium on the mineralization was qualitatively analyzed by Alizarin Red staining after 8, 16, 21, and 28 days: (a) gross examination (b) quantitative data (*P < 0.01, n = 6). From Kim et al., Biomedical Materials, 8(1):014107, Copyright © 2013. Adapted by permission of IOP Publishing
AFS cells can also contribute to bone formation in vivo. Dupont et al. [29] investigated the effect of human AFS cells on bone formation using a nude rat critical-sized femoral segmental defect model. In this study, poly(ε-caprolactone) (PCL) seeded with human AFS cells showed higher bone ingrowth compared to those receiving acellular scaffolds. Various types of stem cells have been investigated for bone formation; however, there have been only few comparisons. Using this model, human AFS cells were compared to human MSCs for their effect on bone repair. 2D X-ray images and micro-CT quantification of bone volume showed that there were no significant differences between these cell sources (Fig. 10.2).
Fig. 10.2
Structure results from in vivo delivery of stem cell-loaded scaffolds. (a) Micro-CT (upper) and X-ray (lower) images of the best bone formation per group in defects receiving acellular scaffold (left), hMSC-seeded scaffold (center), or hAFS cell-seeded scaffold (right). (b) In vivo bone volume comparison showing no significant differences between groups. From Dupont et al., PNAS, 107(8):3305-10, Copyright © 2013. Adapted by permission of PNAS
In Peister’s study [23], however, direct comparisons were made between AFS cells and MSCs in terms of mineralization potential in 3D environments where cells were cultured on scaffolds constructed of PCL for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed after 5 weeks. In contrast, AFS cells showed an extended duration of mineralization that continued to increase to 15 weeks. The total mineralized content in AFS constructs were five times more than MSC constructs (Fig. 10.3). Based on these findings, it was suggested that MSCs would be a good choice for the clinical application where immediate matrix production is needed while the AFS cells would be more effective when robust mineralization is needed for a longer period of time. This study provided a basis of cell sourcing strategies for efficient treatment depending on clinical needs.
