Int J Stem Cells.  2018 Nov;11(2):216-226. 10.15283/ijsc18019.

Comparative Study on Acellular Dermal Graft Versus Propylene Mesh Both Either Loaded or Unloaded with BM-MSCs in Healing of Skull Bone Defect in Rats: Histological and Immunohistochemical Study

• Elkhateb L
• Zohdy A
• Atalla SS
• Moussa MH
• Hamam GG
• Zahra FA

• KMID: 2429554
• DOI: http://doi.org/10.15283/ijsc18019

Abstract

Bone defect occurs as a consequence of many conditions. Diseased bones don't heal properly and defects in face area need proper bone reconstruction to avoid psychological and social problems. Tissue engineering is an emerging new modality of treatment. We thought to study different methods to fill skull bone defect in rats in order to find the most safe and effective method. So, this study was designed to evaluate the efficacy of acellular dermal graft (ADM) versus propylene mesh both either loaded or unloaded with bone marrow derived mesenchymal stem cells (BM-MSCs) in healing of skull bone defect of a 5 mm diameter. The study included 36 adult male Wistar albino rats that were divided into three groups according to the way of filling skull bone defect. Group I: Ia (sham control), Ib (negative control). Group II: IIa (unseeded propylene), IIb (seeded propylene) and Group III: IIIa (unseeded ADM), IIIb (seeded ADM). The trephine operation was done on the left parietal bone. Specimens were collected four weeks postoperative and processed for H&E, osteopontin immunohistochemistry and scanning electron microscope. Morphometric and statistical analysis were also performed. After studying the results of the experiment, we found that propylene mesh and ADM were suitable scaffolds that could support new bone formation in clavarial bone defect. Healing of skull bone defect was better in rats that received seeded scaffolds more than rats with unseeded scaffolds. The seeded ADM showed significant increase in bone forming activity as confirmed by histomorphometric and statistical results.

Keyword

Bone defect; Propylene mesh; Acellular dermal graft; Histology; Bone marrow derived mesenchymal stem cells; Immunohistochemical

MeSH Terms

Adult
Animals
Bone Marrow
Humans
Immunohistochemistry
Male
Mesenchymal Stromal Cells
Methods
Osteogenesis
Osteopontin
Parietal Bone
Rats*
Skull*
Social Problems
Tissue Engineering
Transplants*
Osteopontin

Figure

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  Fig. 1 (A) The defect after trephine. (B) ADM in the defect. (C~E) Phase contrast photomicrographs of MSCs showing (C) Sub-cultured MSCs on day 5. Most cells are spindle in shape with long cytoplasmic processes having granular cytoplasm with vesicular nuclei. (D&E) Cells of primary culture showing MSCs with positive brownish cytoplasmic immune reaction for CD 105 (↑) and CD44 respectively. (F) Propyelene mesh fibers (↑) in culture media with BM-MSCs (*). (G) H&E of ADM after seeding with BM-MSCs. Irregular collagen fibers in papillary (P) and reticular (R) layers are covered with basement membrane (↑). Inset: Cells (▲) between collagen fibers. ADM: acellular dermal graft, BM-MSCs: bone marrow derived mesenchymal stem cells. Phase contrast photomicrographs [C×200], [D×100], [E×400]. H&E [G×400, inset ×1,000].

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  Fig. 2 (A) Ia: Clavarial bone formed of diploe of compact bone with bone marrow space (BM) in-between. Endosteal surface lined with osteogenic cells (↑) and osteoblasts (↑↑), osteocytes inside their lacunae (O), and basophilic cement lines (▲) are seen between bone lamellae (*). (B~ D) Ib: (B) Bone defect filled with dense vascular (BV) connective tissue, few osteogenic cells (↑) at the margin of the defect, small foci of newly formed bone (↑↑). (C) Richly vascularized (BV) dense connective tissue in bone defect, fibroblasts (↑) between bundles of densely arranged collagen fibers (*). Occasional osteoblasts (▲) on bone surface (B). (D) A blood vessel is surrounded with homogenous acidophilic material (↑), mononuclear cells (▲), lymphocytes (thick arrow) and plasma cells (*). H&E [A, C&D] ×1,000. [B] ×400.

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  Fig. 3 (A) IIa: Thin rim of bone (↑) with attached cells on inner and outer surfaces of defect with bone marrow (BM) inbetween. Inset: margin of defect with osteogenic cells (thick arrow). (B~E) IIb: (B) Irregular immature bone (↑) and BM space near the defect. (C) Highly celluar bone margin, osteocytes in their lacunae, osteoblasts (↑), blood vessels (BV) and irrregulaly formed bone (thick arrow) near defect margin. (D) Bone defect filled with many wide spaces (*) separated by thin septa (↑) and covered with periosteum (P). Area of new bone formation is seen (▲). (E) Newly formed bone trabecula (T), oesteogenic cells (↑), osteocyte lacunae (O), an osteoclast (thick arrow) in its Hawship lacuna and BM cells. H&E [A, C&inset of A] ×400. [B&D] ×100. [E]×1,000.

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  Fig. 4 (A~C) IIIa: (A) Highly cellular edge of defect, osteoblasts (↑), newly formed osteocytes (▲). (B) Highly vascular (BV) dermal graft, bone trabecula (T), osteocyte lacunae (O), osteogenic cells (↑). (C) Center of defect: dense connective tissue (*), newly formed bone (▲) surrounded with osteoblasts (c), osteogenic cells (f), blood vessels (↑). (D&E) IIIb: (D) Center of defect: Bone trabeculae (T) and osteogenic cells (↑). (E) Irregular margin of bone defect (↑) and osteoblasts (▲). Inset: Osteoblasts (▲) and highly cellular center of defect. H&E (A~E) ×400. [Inset of E] ×1,000.

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  Fig. 5 Scanning electron micrographs of sagittal section of rat skull bone. (A&B) Ia: (A) Clavarial bone (C) enclosing bone marrow space (BM) is lined with flat endosteal cells (E). (B) Cuboidal bone cells (↑) on the surface of bone trabeculae (T). (C) Ib: Irregularly arranged collagen fibers (↑). Cells (▲) with long processes are attached to collagen fibers and RBCs (*) are seen inbetween. (D&E) IIa: (D) Fibers (*) and cells (↑) filling areas between propylene fibers (P). (C) Calvarial bone. (E) Regularly arranged fibers (*). Flattened cells (thick arrow) and cuboidal cells (▲) with cytoplasmic processes (↑) are seen between these fibers. SEM [A] ×1,000. [B, C&E] ×4,000. [D] ×200.

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  Fig. 6 Scanning electron micrographs of sagittal section of rat cranial vault at the defect area. (A&B) IIb: (A) A fiber of propylene mesh (P), surrounnded by irregular connective tissue filling the defect. Newly formed bone trabeculae can be seen at its periphery (↑). (B) Irregular fibers (↑), interconnected cells (▲) and irregular bone trabeculae (T) are seen between propylene fibers (P). (C) IIIa: Newly formed bone trabeculae (T) and osteocytes (O) inside their lacunae (L) can be seen with their processes (↑). (D) IIIb: The defect is filled with connective tissue. Irregular bone trabecula (T) and cells (↑) with interconnected processes can be seen between the trabeculae. SEM [A] ×200. [B, C&D] ×4,000.

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  Fig. 7 (A) Ia: Positive immune reaction at cement lines (↑), in cells lining the endosteum (▲) [from above downwards: osteogenic, osteoclast, osteoblasts. (B) Ib: Minimal expression in cells lining blood vessels (▲), at margin of defect (↑) and dura (D). (C) IIa: Positive reaction in cells at the margin of defect (↑) and on thin rim of bone on the inner and outer surfaces of defect (▲). (D&E) IIb: Positive immune reaction (D) in osteoblasts (▲) and osteocytes (▲), (E) In cells (↑) surrounding newly formed bone marrow space (BM). Inset: in cells invading the septa that separate bone marrow spaces. (F) IIIa: positive reaction in cells at defect margin (↑), in extracellular matrix (▲). Clavarial bone (B). (G&H) IIIb: Intense reaction in (G): extracellular matrix, osteoblasts (▲), osteocytes (▲), cements lines (↑), (H) in the center of the defect in cells (↑) and extracellular matrix (*). Anti osteopontin antibody [A~F&its inset, G&H] ×400.

Reference

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