TABLE 4.
Preclinical studies using Pullulan scaffolds for bone regeneration in orthotopic sites.
| Author (Year) | Animal model (sex; species); n, number of animals used | Type of defect (defect size) | Conditions (N, number of defects created in total per condition) | Observation time [in day (D), week (W) or month (M)] | Experimental analysis | Results |
|---|---|---|---|---|---|---|
| Hayashi et al. (2009) | Mouse (male, ICR), n = NS | Calvaria implantation (without defect) | 1: Pullulan (N=NS) | 14 D | Micro-CT (BV in mm3) | No significant difference between groups |
| 2: Pullulan + BMP-2 (0.1 µg) (N=NS) | ||||||
| Ø: 4.6 mm | 3: Pullulan + BMP-2 (1 µg) (N=NS) | |||||
| H: NS | 4: Pullulan + BMP-2 (1.5 µg) (N=NS) | |||||
| 4: Pullulan + BMP-2 (2 µg) (N=NS) | HE staining; Von Kossa staining (qualitative analysis) | Pullulan + BMP-2 stimulation of osteoblast activity to form new bone | ||||
| Mouse (male, ICR), n = NS | Calvaria | 1: Empty (N=NS) | 4 W | Micro-CT (qualitative analysis) | Pullulan alone failed to repair bone defect | |
| Ø: 4.6 mm | 2: Pullulan (N=NS) | Pullulan + BMP-2 (2 µg) full repair of bone defect | ||||
| H: NS | 3: Pullulan + BMP-2 (1 µg) (N=NS) | |||||
| 4: Pullulan + BMP-2 (2 µg) (N=NS) | HE staining (qualitative analysis) | New bone formation at the edges of the defect for Pullulan + BMP-2 groups | ||||
| Miyahara et al. (2011) | Rat (NS, Wistar), n = 36 | Calvaria | 1: Empty (N = 24) | 2 W | Micro-CT (BV in mm3) | At 4W: Pullulan membrane * > Collagen membrane |
| Ø: 5 mm | 2: Collagen membrane (N = 24) | 4 W | HE staining (qualitative analysis) | Collagen membrane: immature bone synthesized on both sides of the membrane | ||
| H: NS | 3: Pullulan membrane (N = 24) | 8 W | Pullulan membrane: Mature bone synthesized; regeneration occurred under the membrane | |||
| Fujioka-Kobayashi et al. (2012) | Mouse (NS, C57BL/6N), n = 43 | Calvaria | 1: Pullulan + PBS (N = 11) | 0 W | Micro-CT (Bone healing in %) | At 8W: Pullulan + FGF18 + BMP-2 * > Pullulan + BMP-2 |
| Ø: 3 mm | 2: Pullulan + FGF18 (N = 10) | 1 W | ||||
| H: 5 mm | 3: Pullulan + BMP-2 (N = 11) | 2 W | ||||
| 4: Pullulan + FGF18 + BMP-2 (N = 11) | 3 W | Alizarin Red and Calcein staining (qualitative analysis) | Newly bone synthesis in the defect in Pullulan + BMP-2 and Pullulan + FGF18 + BMP-2 groups | |||
| 6 W | ||||||
| 8 W | ||||||
| Mouse (NS, Osx1-GFP::Cre/B26B), n = 6 | Calvaria | 1: Pullulan-Rh + PBS (N = 3) | 2 W | X-gal staining | Osterix 1 gene activated for the formation of new bone in Pullulan-Rh + FGF18 + BMP-2 group | |
| Ø: 3 mm | 2: Pullulan-Rh + FGF18 + BMP-2 (N = 3) | |||||
| H: NS | ||||||
| Takahata et al. (2015) | Rabbit (female, New Zealand), n = 6 | Ulnar (diaphysis) | 1: α-TCP (N=NS) | 4W | HE staining; Safranin O staining (qualitative analysis) | Mature bone formation for Pullulan + β-TCP group |
| L: 10 mm | 2: Pullulan + β-TCP (N=NS) | 8W | Fragmented bone ingrowth for α-TCP group | |||
| Mouse (female, C57BL/6J), n = 13 | Femoral (Intramedular injection) Size: NA | 1: Empty (N = 4) | 4W | Micro-CT (BMD in mg/cm3) | Pullulan + β-TCP * > Empty | |
| 2: Pullulan (N = 4) | ||||||
| 3: Pullulan + β-TCP (N = 4) | ||||||
| 1: Empty (N = 3) | 2 W | HE staining (qualitative analysis) | At 5W: bone formation began for Pullulan + β-TCP group | |||
| 2: Pullulan (N = 3) | 5 W | |||||
| 3: Pullulan + β-TCP (N = 3) | 8 W | |||||
| Pig (female, NS), n = 4 | Vertebral body Size: 1 cm2 | 1: Empty (N=NS) | 8 W | Micro-CT (CT number in Hounsfield Unit) | Pullulan + β-TCP ** > Empty | |
| 2: α-TCP (N=NS) | ||||||
| 3: Pullulan + β-TCP (N=NS) | ||||||
| 1: α-TCP (N=NS) | 4 W | HE staining (qualitative analysis) | At 4W: bone defect recovered by bone healing for Pullulan + β-TCP group whereas α-TCP group demonstrated fragmented bone ingrowth | |||
| 2: Pullulan + β-TCP (N=NS) | 8 W | |||||
| Charoenlarp et al. (2018) | Mouse (male, C57BL/6N), n = 6–10 per condition | Calvaria | 1: Pullulan (air) + PBS (N=NS) | 0 W | Micro-CT (% of healing) | At 8W: Pullulan (cross-linked RGD) + BMP-2 + FGF18 * > Pullulan (freeze-dried) + BMP-2 + FGF18 |
| Ø: 3 mm | 2: Pullulan (freeze-dried) + PBS (N=NS) | 1 W | ||||
| H: NS | 3: Pullulan (cross-linked RGD) + PBS (N=NS) | 2 W | ||||
| 4: Pullulan (air) + BMP-2 + FGF18 (N=NS) | 3 W | |||||
| 5: Pullulan (freeze-dried) + BMP-2 + FGF18 (N=NS) | 4 W | |||||
| 6: Pullulan (cross-linked RGD) + BMP-2 + FGF18 (N=NS) | 6 W | |||||
| 8 W | Modified Tetrachrome staining (qualitative analysis) | Whether Pullulan was cross-linked with RGD peptides or freeze-dried and combined with BMP-2 and FGF18, it showed trabecular bone formation with calcified bone fragmented | ||||
| Mouse (male, ICR), n = 3–4 per condition | Calvaria | 1: Pullulan (cross-linked RGD) + PBS (N=NS) | 1 W | Modified Tetrachrome staining (qualitative analysis) | Presence of osteoblasts in Pullulan (cross-linked RGD) + BMP-2 + FGF18 and bone regeneration begun under the nanogel | |
| Ø: 3 mm | 2: Pullulan (cross-linked RGD) + BMP-2 + FGF18 (N=NS) | |||||
| H: NS | ||||||
| Popescu et al. (2019) | Rat (male, Wistar), n = 30 | Femoral (diaphysis, unicortical) | 1: Alginate/Pullulan (N=NS) | 0 D | MRI (Bone defect width in µm) | All groups showed a decrease in bone defect size but it was faster for Alginate/Pullulan/1.5CuBG group (No statistical comparison done) |
| 2: Alginate/Pullulan + β-TCP/HA (N=NS) | 5 D | |||||
| 3: Alginate/Pullulan/0.5CuBG (N=NS) | 28 D | |||||
| W: 2 mm | 4: Alginate/Pullulan/1.5CuBG (N=NS) | 0 D | HE staining (qualitative analysis) | Alginate/Pullulan/0.5 or 1.5CuBG showed osteoid tissue formation with parallel organized fibers | ||
| 5: Alginate/Pullulan/BG (N=NS) | 35 D |
BG, bioglass; BMD, bone mineral density; BMP, bone morphogenic protein; BV, bone volume; β-TCP, beta-tricalcium phosphate; H, height; HA, hydroxyapatite; HE, hematoxylin and eosin; L, length; Micro-CT, micro-computed tomography; NA, not applicable; NaCl sodium chloride; NS, not specified; PBS, phosphate buffer saline; Rh rhodamine; W, width; X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside; * p < 0.05; ** p < 0.01; *** p < 0.001; ø, diameter.