We determined the origin of new cartilage and new bone induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) at the site of cricoid cartilage defects in rabbits randomly divided into eight groups. The cricoid cartilage was split vertically along the anterior midline and a strip was excised from the anterior part of the cricoid cartilage in all rabbits. The perichondrium from the anterior part of the cricoid cartilage was trimmed off in four groups; two groups treated with rhBMP-2 and two control groups. In four other groups, the anterior perichondrium was detached and used as a flap with two groups treated with rhBMP-2 and two groups serving as controls. The rabbits were killed 1 week or 4 weeks after surgery. The larynges were removed, fixed and sectioned, and the sections were stained for light microscopy using various cytochemical and immunological techniques. New cartilage was only present close to the host perichondrium adherent to cricoid cartilage in rabbits treated with rhBMP-2. New bone was present 4 weeks after surgery, although calcified matrix and alkaline phosphatase activity could be detected at the site of cricoid defects as early as 1 week after surgery. The cell proliferation marker Ki-67 was strongly expressed in granulation tissue and bone marrow, and it was moderately expressed in muscles adjacent to the cricoid cartilage in rhBMP-2-treated specimens. BMP receptors were strongly expressed in cartilage and moderately expressed in adjacent muscles. We conclude that new cartilage originates from the mesenchymal progenitor cells of host perichondrium adherent to cricoid cartilage in rabbits treated with rhBMP-2. New bone may originate from local muscle.

OBJECTIVE:The study evaluated the possible differences in the repair of cricoid cartilage defects treated with recombinant human BMP-2 in young and adult rabbits. METHODS:A cricoid defect rabbit model was used. Thirty rabbits were randomly divided into eight groups. Two groups of young rabbits and two groups of adult rabbits were treated with rhBMP-2 delivered on an absorbable collagen sponge, while the other two groups of young rabbits and two groups of adult rabbits were used as controls. The rabbits were killed at 1 week or 4 weeks after surgery. A histomorphometric analysis and an evaluation of the expression of collagen types I, II, and X, and proliferating cell nuclear antigen as well as a study of distribution of calcified matrix, were performed. RESULTS:rhBMP-2 induced a marked chondrogenesis in both experimental age groups. However, in young rabbits the newly formed cartilage appeared more elongate, and the length of perichondrium involved was greater. The host cricoid cartilage of adult rabbits was calcified in large areas and displayed a strong matrix expression of collagen type X as well as collagen type I in the perichondrium, compared to the cricoid of young rabbits. In spite of these differences no immunohistochemical differences were found in the newly formed cartilage of both age groups treated with rhBMP-2. The cricoid cartilage defect was filled with new bone at 4 weeks in both age groups treated with rhBMP-2. New bone tissue had a well-defined trabecular structure. CONCLUSIONS:rhBMP-2 triggers appositional cartilage growth from the cricoid perichondrium of young rabbits more easily than from that of adult rabbits. The new bone induced by rhBMP-2 showed a similar immunohistochemical and morphological pattern in both age groups of rabbits.

Objective Bone morphogenetic protein-2 offers potential benefits for cartilage regeneration. We investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the regeneration of laryngeal cartilage and respiratory epithelium in a rabbit model. Material and Methods We used a cricoid defect rabbit model. Twenty-four rabbits were randomly divided into four equal groups. Two groups were treated with 5 μg of rhBMP-2 delivered on an absorbable collagen sponge and the other two groups were used as controls. One group of treated rabbits and one group of control rabbits were euthanized 1 week after surgery, while the others were euthanized 4 weeks after surgery. The healing pattern of the laryngeal wound was evaluated by means of histomorphometry. Results Regeneration of both the epithelial layer and cartilage was significantly better in rabbits treated with rhBMP-2. Four weeks after surgery, the cricoid cartilage defect was completely repaired by new cartilage and new bone in rabbits treated with rhBMP-2. Furthermore, the lining respiratory epithelium healed more rapidly in treated rabbits. Conclusion rhBMP-2, delivered via an absorbable collagen sponge, induces complete regeneration and repair of rabbit cricoid cartilage defects. It also induces faster relining and regeneration of airway epithelium than in control rabbits.

We examined the structural characteristics of repair tissue induced by recombinant human bone morphogenetic protein‐2 in a rabbit model of laryngotracheal reconstruction. Twenty‐four New Zealand White rabbits were randomly divided into four groups of six rabbits. Two groups were treated with recombinant human bone morphogenetic protein‐2 delivered on an absorbable collagen sponge, while two groups were used as controls. Rabbits were euthanized at 1 and 4 weeks after surgery. The larynx was removed, fixed, and sectioned. The sections were stained with hematoxylin‐eosin, safranine O/fast green, and immunostained with an antibody for tissue inhibitor of metalloproteinases‐1. In rabbits treated with bone morphogenetic protein‐2, the defects were filled with new cartilage and bone at 4 weeks after surgery. There were no discontinuities or gaps at the margins of the cartilage defects. Proteoglycans were synthesized in new cartilage in rabbits treated with bone morphogenetic protein‐2, and were present 4 weeks after surgery. The general aspects of the vascular pattern and the pattern of tissue inhibitor of metalloproteinases‐1 expression were similar in control and treated rabbits, both 1 week and 4 weeks after surgery. The repair tissue induced by recombinant human bone morphogenetic protein‐2 consisted of new cartilage and bone perfectly integrated with host tissue at the site of the cricoid cartilage defects. This new cartilage was able to mature and produce proteoglycans.

Apoptosis in normal meatal skin, cholesteatoma and squamous cell carcinoma (SCC) of the ear was investigated by using morphological analysis and in-situ specific labelling of fragmented DNA. In meatal skin, apoptotic cells were localized mainly in the granular layers, but were not so restricted in cholesteatoma, while in SCC they were even more dispersed. The apoptotic index (AI) was low (1.59 +/- 0.10 SEM) in normal skin. It was increased in cholesteatoma (2.09 +/- 0.11), and was intermediate in SCC (1.72 +/- 0.14). By contrast, the mitotic index (MI) increased from 0.19 +/- 0.02 in normal skin, to 0.25 +/- 0.01 in cholesteatoma and to 0.25 +/- 0.02 in SCC. Our findings indicate that apoptosis is involved in the epithelial homeostasis of meatal skin, cholesteatoma and SCC of the ear. The hyperproliferation of epithelial cells in cholesteatoma is counteracted by an increased apoptosis rate, while in SCC the increased cell proliferation without a compensatory increase in apoptosis may be associated with the malignant transformation.

Lectin expression during wound healing of the rabbit sinus mucosa was examined. Positive UEA-I staining was evident on squamous or cuboidal as well as columnar regenerating epithelial cells (RE cells). PNA staining of columnar RE cells first became evident after neuraminidase treatment, while squamous or cuboidal RE cells stained positively with PNA alone. Fucosylation within RE cells thus occurred from a relatively early period, and sialylation followed at later stages. Ingrowing epithelial cells of early polyp formation stained negatively with UEA-I, indicating that unfucosylated RE cells may represent aberrant cellular behavior. We concluded that these patterns of lectin staining indicate a functional maturation as well as an integration of regenerating mucosa.

To evaluate the pattern of goblet cell differentiation in sinus mucosa in response to external stimuli, New Zealand White rabbits were subjected to either experimental sinusitis or topical capsalcin application. Sinus mucosa was examined by light microscopy after serial sectioning, whole-mount preparation or immunohistochemistry. The mucosa was also examined by electron microscopy after perfusion fixation or high-pressure freezing. While goblet cells were normally very scarce in the healthy rabbit maxillary sinus mucosa, such cells were frequent after experimental sinusitis or topical capsaicin application. The process of goblet cell differentiation seems to follow a sequential path where serous secretory cells start to produce an increasing amount of mucous granules which appear electron lucent after conventional fixation. Parallel to this shift in secretion production, the cell assumes a bulging appearance after conventional fixation. It is concluded that newly formed goblet cells are recruited from intermediate secretory cell stages rather than from ciliated cells.

Lectin expression in both normal and inflamed sinus mucosa in rabbit was investigated histochemically. A different staining pattern was observed in the inflamed mucosa, i.e. the degree of staining reactivity with Canavalia ensiformis (ConA) decreased, whereas staining with Ulex europaeus agglutinin-I (UEA-I), Arachis hypoga (PNA) with neuraminidase pretreatment and Triticum vulgaris (WGA) intensified, indicating an enhanced fucosylation as well as sialylation. Goblet cells were stained with UEA-I, WGA, PNA and PNA with neuraminidase pretreatment, but scarcely with ConA. Positive PNA staining of basal cells and epithelial secretory granules was observed in the inflamed mucosa, especially close to areas where goblet cell development was prolific. It was therefore assumed that basal cells can differentiate into goblet cells-through epithelial secretory cells accompanied by sialylation and/or sulphonation of their mucins. It is concluded that the changes in glycoconjugate expression as well as goblet cell development in the inflamed mucosa are of importance for both local host resistance and defence mechanisms against microorganisms during the early stages of the inflammatory process.

The effect of airway infection on neurogenic inflammation is not known. The present study examines the effect of Mycoplasma pulmonis infection on the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) in the trigeminal ganglion and in the mucosa of the nose and trachea in rats. We compared germ-free (GF), conventionally raised (CV) and specific pathogen-free (SPF) rats. The concentrations of SP and CGRP in the nasal mucosa were assessed with immunohistochemistry, and their prohormonal transcripts in the trigeminal ganglion were assessed with Northern blot. Mucosa was also processed for light microscopy and electron microscopy. SP-like immunoreactivity was greater in the nasal mucosa of infected animals than in uninfected controls. CGRP-like immunoreactivity was greater in the nasal septum, but not in the nasal turbinate, of infected than uninfected animals. In contrast, no change was evident in the expression levels of the prohormonal transcripts in the trigeminal ganglion. Infected nasal and tracheal mucosa was oedematous and locally infiltrated with inflammatory cells. In the nose of uninfected GF rats, subepithelial lymphoid aggregations were scarce and appeared inactive. We conclude that Mycoplasma pulmonis infection results in increased immunoreactivity of substance P, probably within nerves. There was no clear evidence of increased synthesis of the precursors of substance P and calcitonin gene-related peptide.

In order to study morphological effects on the nasal and sinus mucosa, New Zealand White rabbits underwent either unselective, regional sectioning of sensory and parasympathetic nerve branches or topical treatment of the mucosa with capsaicin. Ten days after treatment, mucosal specimens were analyzed by light and electron microscopy. Immunohistochemistry was used to evaluate neuropeptides present, in particular substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and neuropeptide Y. In surgically denervated rabbits, mucosal glands were found to be enlarged and contained an increased number of zymogen granules having a bipartite substructure. Topical capsaicin application caused localized epithelial changes in the sinus mucosa and maxilloturbinal region of the nose, including clotting of cilia and an increased number of goblet cells. Reduced amounts of all neuropeptides investigated were found in the surgically denervated animals, while topical capsaicin treatment had only marginal effects on the mucosal neuropeptide content. The morphological changes observed after surgical denervation suggest an imbalance between neural stimulation and secretory capacity of the mucosal glands. These findings could explain the difference in clinical effect noted between sectioning of the vidian nerve and topical treatment with capsaicin in patients with perennial rhinitis.

To study the possible autocrine growth stimulation of cholesteatoma epithelium, the expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in middle ear cholesteatoma was investigated by immunohistochemical staining techniques. Twenty cholesteatoma samples obtained at operation and six normal skin specimens collected from the external ear canal were used in the study. Immunostaining for TGF-alpha showed a diffuse cytoplasmic staining pattern. It was weakly expressed in the basal layer of the normal epidermal epithelium but was more strongly expressed in all cholesteatoma specimens. EGFR showed a dot-like/diffuse cytoplasmic and cell membrane staining pattern. EGFR-positive cells were seen in the basal layer of normal epidermis. In the cholesteatoma specimens, expression of EGFR was not only confined to the basal layer but persisted into the upper layers of the epithelium. Our findings indicate that an autocrine growth stimulation by TGF-alpha and EGFR may contribute to the unrestrained growth of cholesteatoma epithelium in the middle ear cavity.

The expression of epithelial markers (cytokeratins, Filaggrin. BerEp4 and EMA), collagen IV and Ki67 was studied immunohistochemically in cholesteatoma and compared with that in epidermis of meatal skin, squamous epithelium of eardrum and simple epithelium of middle ear mucosa. MNF116 (cytokeratin 10, 17, 18) stained the full layer of normal epithelium and all cholesteatoma specimens. CK10 and Filaggrin were expressed in the upper layer of epidermis but more diffusely in cholesteatoma. BerEp4 was found in the basal layer of normal epithelium but was detected in most epithelial cells in cholesteatoma matrix. Variability was observed in EMA and CK14 immunostaining. Collagen IV was localized in the basement membrane of normal epithelium with a continuously staining pattern, an observation also made in the cholesteatomas studied. However, in one of these small areas the basement membrane was not stained with collagen IV. Ki67 was expressed in nuclei of the cells in the basal layer of normal epithelium but extended to epithelial cells in the upper layers of cholesteatoma matrix. The results of the present study indicate that the expression pattern of epithelial markers in cholesteatoma corresponds to that in normal epidermis. The increasing expression of BerEp4 and Ki67 confirms the hyperproliferative nature of cholesteatoma. Whether or not the lack of expression of collagen IV in one of the cholesteatomas reflects a true degradation of the basement membrane needs further investigation in extended materials.

Rabbit maxillary sinuses were inoculated with Streptococcus pneumoniae and Bacteroides fragilis, and the histologic response in the sinus mucosa was observed over a 12-week period. An increased height of the cylindric cells and hyperplasia of the basal cells were frequent findings irrespective of the pathogen inoculated. The disease was found to influence the character of the secretory product from epithelial secretory cells and to degranulate the subepithelial glands. Ciliary loss was a transitional finding. A reduction in the number of mitochondria, the occurrence of deformed short microvilli, and cytoplasmic blebbing were seen in the cells devoid of normal cilia. It is inferred from this study that pneumococcal sinusitis in rabbits is a self-limiting process, and the mucosal sequelae of the acute infection are persisting goblet cells, slight focal fibrosis, and edema. Inoculation with B fragilis produces a chronic inflammatory process with infiltration of mononuclear cells, luminal dilatation of the glands exhibiting zymogen granule depletion, and an increased thickness of the whole mucosal layer.

Unilateral maxillary sinusitis was experimentally induced in New Zealand White rabbits with Streptococcus pneumoniae serotype 3, Bacteroides fragilis NCTC 9343, and Staphylococcus aureus V8. In another group of rabbits, sinusitis was induced by blocking of the sinus ostium only. Bacteriologic and light microscopic analysis was performed after 5 days to 1 month. Granulation-like polyps developed after deep mucosal inflammatory trauma initiating fibroblast proliferation, angiogenesis, and epithelial migration to cover the polyp. In regions of a more superficial trauma-characterized by epithelial desquamation and fibroblast growth-proliferation and differentiation of basal cells resulted in the formation of microcavities dissecting off edematous polyps. Polyps could be found in all sinusitis groups, irrespective of inducing agent. The cellular events of polyp formation appear to be the result of a continuous inflammatory reaction and are not directly related to the presence of a certain microorganism. Instead, the potential of any microorganism to induce a deep mucosal trauma or epithelial desquamation seems essential for its ability to initiate polyp formation.

A rapid regeneration of the epithelium takes place in the maxillary sinus in rabbits after experimental operative removal of the mucosa. Two weeks postoperatively the previously denuded areas have reepithelialized. The subepithelial glands, however, do not seem to regenerate. The normal sinus mucosa contains numerous serous glands in the lamina propria, but in the regenerated mucosa these glands are replaced by dense connective tissue. Atypical glands and polyp formations are sometimes encountered, but goblet cells are sparse. Furthermore, the sinus cavity on the operated side is reduced in size compared with the nonoperated side because of fibrosis and periosteal reactions including bone degradation and neogenesis. This study indicates that although the mucosa is reepithelialized within 2 weeks, the regeneration of the lamina propria is incomplete, and reactive cellular processes such as bone remodeling, fibroblast proliferation, and formation of polyps and “atypical glands” are characteristic of regenerating mucosa.

Unilateral maxillary sinusitis was experimentally induced in New Zealand White rabbits with Streptococcus pneumoniae serotype 3, Bacteroides fragilis NCTC 9343, and Staphylococcus aureus V8 in order to study possible differences in the inflammatory response of the sinus and nasal mucosa at different time-intervals during a 12-week period of infection. The initial sinus mucosal response, most pronounced in pneumococcal sinusitis, was characterized by leukocytosis, epithelial desquamation, and squamous cell metaplasia. Tissue reactions at later intervals included fibrosis of lamina propria, gland involution, polyp formation, and bone remodelling, and were most pronounced in S. aureus and B. fragilis sinusitis. The nasal mucosa was altered with a redistribution of goblet cells, development of polyps in the ethmoidal region, involution of Bowman's glands and locally, areas of degenerated olfactory sensory epithelium. These findings endorse that the degree of local pathology depends on the infecting microorganism's specific pathogenetic factors. However, local tissue factors guiding the cellular inflammatory proliferative and regenerative processes are also of fundamental importance for the type of pathological changes occurring in an infected nasal or sinus mucosa.