Bone Regeneration Using the Freshly Isolated Autologous Stromal Vascular Fraction of Adipose Tissue in Combination With Calcium Phosphate Ceramics

Abstract

: In patients undergoing maxillary sinus floor elevation (MSFE) for dental implant placement, bone substitutes are currently evaluated as alternatives for autologous bone. However, bone substitutes have only osteoconductive properties and lack osteoinductive potential. Therefore, this phase I study evaluated the potential additive effect on bone regeneration by the addition of freshly isolated, autologous but heterologous stromal vascular fraction (SVF), which is highly enriched with adipose stromal/stem cells when compared with native adipose tissue. From 10 patients, SVF was procured using automatic processing, seeded on either β-tricalcium phosphate (n = 5) or biphasic calcium phosphate carriers (n = 5), and used for MSFE in a one-step surgical procedure. Primary objectives were feasibility and safety. The secondary objective was efficacy, evaluated by using biopsies of the augmented area taken 6 months postoperatively, concomitant with dental implant placement. Biopsies were assessed for bone, graft, and osteoid volumes. No adverse effects were reported during the procedure or follow-up (≥3 years). Bone and osteoid percentages were higher in study biopsies (SVF supplemented) than in control biopsies (ceramic only on contralateral side), in particular in β-tricalcium phosphate-treated patients. Paired analysis on the six bilaterally treated patients revealed markedly higher bone and osteoid volumes using microcomputed tomography or histomorphometric evaluations, demonstrating an additive effect of SVF supplementation, independent of the bone substitute. This study demonstrated for the first time the feasibility, safety, and potential efficacy of SVF seeded on bone substitutes for MSFE, providing the first step toward a novel treatment concept that might offer broad potential for SVF-based regenerative medicine applications.

Significance: This is the first-in-human study using freshly isolated, autologous adipose stem cell preparations (the stromal vascular fraction [SVF] of adipose tissue) applied in a one-step surgical procedure with calcium phosphate ceramics (CaP) to increase maxillary bone height for dental implantations. All 10 patients received CaP plus SVF on one side, whereas bilaterally treated patients (6 of 10) received CaP only on the opposite side. This allowed intrapatient evaluation of the potential added value of SVF supplementation, assessed in biopsies obtained after 6 months. Feasibility, safety, and potential efficacy of SVF for bone regeneration were demonstrated, showing high potential for this novel concept.

Keywords: Adipose stem cells; Bone regeneration; Calcium phosphates; Clinical study; Maxillary sinus floor elevation; Regenerative medicine.

©AlphaMed Press.

Figures

Figure 1.

Schematic overview of one-step surgical procedure, maxillary sinus floor elevation, and biopsy analysis. (A): The plastic surgeon starts harvesting adipose tissue by liposuction. (B): The adipose tissue and liposuction fluid is collected in syringes. (C): The filled syringes are transferred into a Celution 800/CRS system. This device washes, digests, and centrifugates the adipose tissue to obtain the fresh stromal vascular fraction containing the adipose stem cells. After isolation of the stromal vascular fraction, cells can be shortly stimulated with the growth factors before seeding the stimulated cells onto a carrier material. (D): The freshly isolated adipose stem cells are seeded onto the calcium phosphate carrier. Unattached cells are washed off (“stam”=stem cells). (E): During the short attachment period of the cells (30 minutes), the patient is prepared for the maxillary sinus floor elevation procedure via a lateral approach. After reflection of the mucoperiosteal flap, a bony window is created in the lateral wall of the maxillary sinus and carefully moved and rotated medially toward the maxillary sinus, after dissection of the maxillary sinus mucosa (trap-door technique). (F): The tissue-engineered construct is inserted immediately into the patient, and the space created is filled with the bone substitute combined with the adipose stem cells. (G): Finally, the wound is closed. (H): After 6 months, bone biopsies are taken by using a hollow burr, and dental implants are placed (I). (J): Bone biopsies are removed from the hollow burr and analyzed by using micro-computed tomography (micro-CT) evaluation (K) and histomorphometry (L).

Figure 2.

Cell seeding and attachment. (A): A representative image of calcium phosphate material seeded with stromal vascular fraction cells and fluorescently stained with 4',6-diamidino-2-phenylindole to visualize attached cells (blue) by fluorescence microscopy. Scale bar represents 200 µm. (B): Scanning electron microscope image demonstrating attached cells on the calcium phosphate material (2500× magnification). Scale bar represents 10 µm. The inset is a representative scanning electron microscope image of the calcium phosphate granules at a lower magnification (×100 magnification). Scale bar represents 200 µm.

Figure 3.

Micro-computed tomography analysis of selected bone biopsies taken from control sides without stem cells (white bars; n = 3) and study sides with stem cells (black bars; n = 5) from patients treated with β-tricalcium phosphate (A, B, E, F) or biphasic calcium phosphates (C, D, G, H). (A, C): Percentage mineralized BV/TV. (B, D): Percentage BV/TV in pooled native bone as well as per ROI. (E, G): Percentage GV/TV. (F, H): Percentage GV/TV in pooled native bone as well as per ROI. Data were shown only for ROIs n ≥ 3. Abbreviations: β-TCP, β-tricalcium phosphate; BCP, biphasic calcium phosphates; BV, bone volume; GV, graft volume; ROI, region of interest; TV, total volume.

Figure 4.

Histomorphometric analysis of selected bone biopsies taken from control sides without stem cells (white bars; n = 3), and study sides with stem cells (black bars; n = 5) from patients treated with β-TCP (A, B, E, F, I, J) or BCP (C, D, G, H, K, L). (A, C): Percentage mineralized BV/TV. (B, D): Percentage BV/TV in pooled native bone as well as per ROI. (E, G): Percentage GV/TV. (F, H): Percentage GV/TV in pooled native bone as well as per ROI. (I, K): Percentage unmineralized OV/TV. (J, L): Percentage OV/TV in pooled native bone as well as per ROI. Data were shown only for ROIs n ≥ 3. Abbreviations: BCP, biphasic calcium phosphates; BV, bone volume; GV, graft volume; OV, osteoid volume; ROI, region of interest; β-TCP, β-tricalcium phosphate; TV, total volume.

Figure 5.

Paired micro-computed tomography (A, B) and histomorphometric analysis (C–E) of selected paired bone biopsies taken from control sides without stem cells and study sides with stem cells within the same patient following a “split-mouth design” (n = 6). A paired one-tailed Student’s t test was performed to assess whether bone and osteoid volumes were higher and graft volumes lower at the study sides than at the control sides. ∗Significantly different from control, p < .05. Abbreviations: BV, bone volume; CT, computerized tomography; GV, graft volume; OV, osteoid volume; TV, total volume.