Acid bone lysates reduce bone regeneration in rat calvaria defects

Abstract

Acid bone lysates (ABLs) represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone-derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of ABLs adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats, a standardized 5-mm-diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum-free media or ABLs followed by lyophilization. After a healing period of 4 weeks, micro-computed tomography (μCT) and histological analyses by means of undecalcified thin ground sections were performed. μCT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to ABL group, 29.8% (confidence interval [CI]: 17.7-50.3) versus 5.6% (CI: 1.0-29.8, p = .03), respectively. Moreover, we found significantly more absolute bone volume (BV) in the control group when compared to ABL group, 0.59 mm³ (CI: 0.27-1.25) versus 0.07 mm³ (CI: 0.06-0.59, p = .04), respectively. Histomorphometry confirmed these findings with a relative BV in the central compartment of 14.1% (CI: 8.4-20.6) versus 5.6% (CI: 3.4-7.9, p = .004), respectively. These findings indicate that bone-derived growth factors contained in ABLs are able to attenuate bone regeneration within collagen membranes.

Keywords: bone allograft; bone grafts; bone regeneration; growth factors.

FIGURE 1

Micro‐CT overview of the defect anatomy, bone in contact with host bone and bony islands after 4 weeks of healing. Rat calvaria defects were treated with collagen membranes either soaked in (a, c, e) serum‐free medium, or (b, d, f) in acid bone lysates (ABL). Micro‐CT pictures representing the samples with (a, b) minimum, (c, d) median, and (e, f) maximum bone volume based on quantitative analysis

FIGURE 2

Acid bone lysate (ABL) reduces bone regeneration in the calvaria defect. Quantitative analysis of (a) bone defect coverage and (b) bone volume in the region of interest (the inner 4 mm diameter of a 5 mm defect). Statistical analysis was based on the data observed with the μCT analysis. The two groups were compared with Mann–Whitney U test. p‐values are indicated

FIGURE 3

Histological overview of the defect anatomy after 4 weeks of healing. Rat calvaria defects were treated with native collagen membranes either soaked in serum‐free medium (a, c, e), or in acid bone lysate (ABL) (b, d, f). Histological pictures representing the samples with minimum (a, b), median (c, d), and maximum (e, f) bone volume based on quantitative analysis. The local host calvaria bone demarcates the defect borders and appears in light purple. The newly formed bone stained in dark purple appears in the spongy part of the collagen membranes

FIGURE 4

The effect of acid bone lysates is restricted to the central compartment. (a) Histomorphometry on bone volume per tissue volume BV/TV (%) was performed at three regions of interest (ROI); (b, Red ROI) the central compartment within defect margins, (c, yellow ROI) the adjacent ectocranial compartments, and (d, green ROI) the outer compartment on the surface of the host cortical bone. The groups were compared with Mann–Whitney U test. p‐values are indicated

FIGURE 5

Detailed overview on the new bone in the control group (CG) and in the acid bone lysate (ABL) group. Note the characteristic features of immature woven bone indicated by the intense purple stain and the large osteocyte lacunae. The dense art of the membrane is visible in the upper part of control group also showing that new bone grows on the spongy part of the collagen membrane