J Periodontal Implant Sci.  2016 Aug;46(4):220-233. 10.5051/jpis.2016.46.4.220.

“Over-inlay” block graft and differential morphometry: a novel block graft model to study bone regeneration and host-to-graft interfaces in rats

• Ghiacci G
• Graiani G
• Ravanetti
• Lumetti S
• Manfredi E
• Galli C
• Cacchioli
• Macaluso G
• Sala R

• KMID: 2350023
• DOI: http://doi.org/10.5051/jpis.2016.46.4.220

Abstract

PURPOSE
The aim of this study was to present new a model that allows the study of the bone healing process, with an emphasis on the biological behavior of different graft-to-host interfaces. A standardized "over-inlay" surgical technique combined with a differential histomorphometric analysis is presented in order to optimize the use of critical-size calvarial defects in pre-clinical testing.
METHODS
Critical-size defects were created into the parietal bone of 8 male Wistar rats. Deproteinized bovine bone (DBBM) blocks were inserted into the defects, so that part of the block was included within the calvarial thickness and part exceeded the calvarial height (an "over-inlay" graft). All animals were sacrificed at 1 or 3 months. Histomorphometric and immunohistochemical evaluation was carried out within distinct regions of interest (ROIs): the areas adjacent to the native bone (BA), the periosteal area (PA) and the central area (CA).
RESULTS
The animals healed without complications. Differential morphometry allowed the examination of the tissue composition within distinct regions: the BA presented consistent amounts of new bone formation (NB), which increased over time (24.53%±1.26% at 1 month; 37.73%±0.39% at 3 months), thus suggesting that this area makes a substantial contribution toward NB. The PA was mainly composed of fibrous tissue (71.16%±8.06% and 78.30%±2.67%, respectively), while the CA showed high amounts of DBBM at both time points (78.30%±2.67% and 74.68%±1.07%, respectively), demonstrating a slow remodeling process. Blood vessels revealed a progressive migration from the interface with native bone toward the central area of the graft. Osterix-positive cells observed at 1 month within the PA suggested that the periosteum was a source of osteoprogenitor elements. Alkaline phosphatase data on matrix deposition confirmed this observation.
CONCLUSIONS
The present model allowed for a standardized investigation of distinct graft-to-host interfaces both at vertically augmented and inlay-augmented sites, thus possibly limiting the number of animals required for pre-clinical investigations.

Keyword

Bone regeneration; Bone transplantation; Histology; Rat; Skull; Surgical procedure

MeSH Terms

Alkaline Phosphatase
Animals
Blood Vessels
Bone Regeneration*
Bone Transplantation
Humans
Male
Osteogenesis
Parietal Bone
Periosteum
Rats*
Rats, Wistar
Skull
Transplants*
Alkaline Phosphatase

Figure

• 

  Figure 1 Surgical procedure for “over-inlay” grafting. (A) Osteotomy on parietal bone created by means of a trephine bur with a 5-mm external diameter (B) Filling of the defects with a cylindrical block graft which partially exceeded calvarial height. Optical camera (Nikon D3x, 24.5 megapixels).

• 

  Figure 2 (A) Scheme of “over-inlay” graft and surrounding host tissues in a sagittal section: grafting material (DBBM), host trabecular bone (TB), host periosteum (P), host cortical bone (CB). Dotted line: distinction between “over” and “inlay” component of the graft. (B) Histological specimen stained with hematoxylin-eosin, showing the typical aspect of “over-inlay” graft. Optical microscopy, original magnification, 2×. (C) Scheme of regions of interest (ROIs) considered for differential histomorphometric and immunohistochemical evaluation: periosteal area (PA), lateral areas adjacent to native host bone (BA), and central area of the defect (CA). New bone formation (NB), fibrous tissue (FT), and residual deproteinized bovine bone (DBBM) were quantified within each of these ROIs, as well as alkaline phosphatase (Alp) and osterix (Osx) expression. (D) Histological specimen stained with hematoxylin-eosin, showing PA, BA, and CA ROIs considered for histomorphometric evaluation. Optical microscopy, original magnification, 2×.

• 

  Figure 3 Representative histological details of lateral areas adjacent to native host bone (BA), periosteal areas (PA), and central areas of the defect (CA) at 1 month and 3 months of healing. Areas of new bone formation (NB), fibrous tissue (FT), and grafting material (DBBM) are marked with letters. Black stars outline periosteum level. Hematoxylin-eosin staining. Optical microscopy, original magnification, 10×.

• 

  Figure 4 (A-C) New bone (NB), fibrous tissue (FT), and deproteinized bovine bone (DBBM) within lateral areas adjacent to native host bone (BA) (A), periosteal areas (PA) (B), and central area of the defect (CA) (C) at 1 and 3 months of healing. Results are expressed as mean percentages/regions of interest (ROIs).

• 

  Figure 5 (A) Blood capillary density within lateral areas adjacent to native host bone (BA), periosteal areas (PA), and central area of the defect (CA) at 1 month and 3 months of healing. Results are reported as number of capillaries (n) over mm2, mean±standard error (SEM). (B) Microscopic image showing blood vessels within PA at 1 month. Vessels are identified by brown of FVIII IHC-staining revealed by DAB. Optical microscopy, scale bar, 10 μm. (C) Density of alkaline phosphatase (Alp) positivity within lateral areas adjacent to BA, PA, and CA at 1 month and 3 months of healing. Results are reported as the number of positive elements (n) over mm2, mean±SEM. (D) Alp positivity visualized with green fluorescence. The blue fluorescence of DAPI identifies nuclei. The image was captured within PA at 1 month. Fluorescence microscopy, scale bar, 50 μm. (E) Density of osterix (Osx)-positive cells within lateral areas adjacent to BA, PA, and CA at 1 month and 3 months of healing. Results are reported as the number of Osx-positive cells (n) over mm2, mean±SEM. a)Statistically significant between PA and indicated groups, P<0.05. (F) Osx-positive cells visualized with green fluorescence. The blue fluorescence of DAPI identifies nuclei. White arrows indicate cells with Osx-positivity. The image was captured within PA at 1 month. Fluorescence microscopy, scale bar 50 μm.

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