Can Collagen Two Help Repair Sinus Cartlidge
Tissue Eng Role A. 2022 October; 19(19-xx): 2201–2214.
Marine Collagen Scaffolds for Nasal Cartilage Repair: Prevention of Nasal Septal Perforations in a New Orthotopic Rat Model Using Tissue Engineering Techniques
Christian Bermueller, MD,1, * Silke Schwarz, MSc, 
1, * Alexander F. Elsaesser, MSc,1 Judith Sewing, MSc,two, iii Nina Baur, MSc,i Achim von Bomhard, MD,ane Marc Scheithauer, MD,ane Holger Notbohm, PhD,ii and Nicole Rotter, Doci
Christian Bermueller
aneDepartment of Otorhinolaryngology, Ulm University Medical Middle, Ulm, Frg.
Silke Schwarz
oneDepartment of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany.
Alexander F. Elsaesser
oneDepartment of Otorhinolaryngology, Ulm University Medical Heart, Ulm, Federal republic of germany.
Judith Sewing
2Institute for Virology and Cell Biology, University of Lübeck, Lübeck, Deutschland.
3CRM, Coastal Research & Management, Kiel, Germany.
Nina Baur
iDepartment of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany.
Achim von Bomhard
1Department of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany.
Marc Scheithauer
1Department of Otorhinolaryngology, Ulm University Medical Middle, Ulm, Federal republic of germany.
Holger Notbohm
2Establish for Virology and Cell Biological science, University of Lübeck, Lübeck, Federal republic of germany.
Nicole Rotter
iDepartment of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany.
Received 2022 Nov 2; Accepted 2022 Apr 16.
Abstract
Autologous grafts are often needed for nasal septum reconstruction. Because they are only available in limited amounts, at that place is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable selection. And then far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. Offset, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. 2nd, nosotros worked toward developing a suitable orthotopic brute model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for one, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce whatever cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such every bit collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a pregnant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent backdrop for cartilage tissue technology. Marine collagen scaffolds are able to foreclose septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable fashion to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.
Introduction
Damage or malformation of cartilaginous facial structures, such every bit the nose or the auricle, may occur as the upshot of trauma, tumor resection, or built defects. Such defects may lead to functional issues, and severe psychosocial strain for the afflicted patient.1,2
The express self-repair capacity2,iii of cartilage necessitates the use of either autologous transplants, such equally rib or auricular cartilage, or constructed materials, such as Gore-Tex or silicone.three The current gold standard for reconstruction of facial cartilage defects is autologous cartilage. In circuitous defects, multistage surgical procedures are required.ii–four Donor-site morbidity is common when large pieces of autologous cartilage are harvested.i,5 Nonetheless, nasal and auricular reconstructions have significant psychosocial benefits in the majority of treated patients.half dozen
The employ of nonresorbable implants oft results in infection and/or extrusion of the implant.seven,8 Thus, there continues to exist demand for culling materials.one,2 Tissue applied science is seen as a promising method for reconstructing auricular and nasal cartilage. Resorbable scaffold materials could be used to adapt cells. The total mechanical stability of the cell-scaffold construct should non alter over fourth dimension, and the mechanical backdrop initially exhibited past the biomaterial should be transferred to the newly synthesized matrix in vivo. A multitude of different natural and synthetic materials take been tested so far.9–17 However, none has proven to exist optimaleighteen–20 for the reconstruction of cartilage in the caput and neck region. This is mainly due to poor mechanical functioning, which is of particular business organisation in the head and neck expanse. Additionally, many of the materials tin cause local inflammatory reactions.
The detail demands of cartilage reconstruction in head and neck surgery crave a specific scaffold design. Start, the structural architecture of the scaffold should mimic the exact shape of the native tissue and should support the attachment, proliferation, and differentiation of the desired cell type.21 Second, the scaffold must be rigid enough to stabilize the reconstructed nasal cartilage until the newly synthesized extracellular matrix (ECM) attains total mechanical stability and function. The cartilaginous septum is responsible for the shape and tension of its surrounding structures (alar cartilages, columella, and nasal back), and these structures in turn enable nasal breathing. Thus, rigidity and stability of the scaffold are of utmost importance.
For many years, collagen has been used as a biomaterial in a multifariousness of connective tissue engineering applications,22 but collagen has also played an important role in other fields of bioengineering.23–29 Collagen has many desirable characteristics for scaffold production and application. Information technology provides fantabulous biocompatibility, low antigenicity, and loftier biodegradability, and collagen's cell binding and signaling properties help to promote cellular processes leading to tissue formation.22–24 For this reason, collagen, which may exist harvested from bovine skin and bone, is regarded as ane of the most favorable materials for bogus ECM replacement.22 Still, considering it is harvested from vertebrate animals, information technology has the disadvantage of potentially carrying transmissible diseases.22,thirty
Nearly marine animals are invertebrates, and so marine collagen is costless of substances that would exist pathogenic to humans.30 Several studies accept demonstrated that invertebrate fibrillar collagens possess the aforementioned characteristics as their homo counterparts.31 Due to their biological properties, new matrices based on marine collagen were recently tested in biomedical applications, such as tissue technology vascular grafts. Such matrices might correspond an alternative source for the production of scaffolds for tissue applied science applications.30,32–34 Marine collagen is harvested past lyophilization of the invertebrate jellyfish species Rhopilema esculentum. Advantages of this collagen source include its reproducible product process, which relies on renewable natural resources, and the absence of whatsoever gamble for disease transmission by viruses or bacteria.30
The other vital part of a tissue-engineered cartilage construct is the cellular component. For clinical applications, access to a suitable source of autologous cells is essential to avert disease transmission35 and to avert a potential immunological rejection acquired past an allogenic donor cell source.36–38 In contempo years, hyaline human nasal septum cartilage has been shown to be useful every bit an alternative autologous cell source because harvesting cartilage from the nasal septum requires minimally invasive surgeries with minimal donor site morbidity.39 Compared with articular and auricular chondrocytes, nasal septum chondrocytes have been reported to display increased cartilage formation and better mechanical stability in the resulting neo-cartilage.sixteen,36,38–forty
Aside from biomaterial and donor cell considerations, the nasal septum has specific immunological and mechanical characteristics. The influence of these characteristics can only be analyzed in detail in a suitable beast model.
The goal of this study was to evaluate the biocompatibility of marine collagen in vitro and in vivo and to determine whether it can be used equally a new biomaterial for nasal septum reconstruction. Nosotros examined the suitability of marine collagen for growing human and rat nasal septal chondrocytes, and nosotros farther aimed to develop a nasal septum-specific immunocompetent animal model to study the furnishings of site-specific local influences on tissue-engineered cartilage.
Materials and Methods
All experiments were performed with the approval of the Regional Ethical Board in Tuebingen, Frg, authoritative decision no. 949/2009.
Marine collagen scaffolds
Triple helical homotrimer marine collagen was harvested from the jellyfish Rhopilema esculentum. All steps were carried out at 4°C–six°C. Cured jellyfish was cut into small-scale pieces, rinsed several times with tap water, and washed for several hours in Milli-Q water until the salinity was below 0.01. After equilibration in 0.5 1000 acetic acrid, the material was homogenized in 0.five M acetic acrid. Enzymatic digestion was performed for approximately 70 h using a pepsin solution containing 10 mg g−1 bones raw cloth (porcine gastric mucosa; Biozym). After centrifugation at 17,000 g for 30 min, the supernatant was neutralized (pH seven.0) and the collagen was precipitated with 17.5% KCl and 0.2 One thousand NaH2POfour. The collagen was obtained by centrifugation for 20 min at 17,000 yard followed by dialysis confronting 0.05% acetic acid. Pepsin remained in the supernatant.
For scaffold product, the harvested collagen was freeze-dried (20 mg mL−i), suspended in 0.05% acetic acid, and injected into a preformed synthetic casting mold. Scaffold material was frozen (at a rate of −0.25°C min−ane) until it reached −20°C before being freeze-stale. Cross-linking was carried out with one-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 0.05/0.xv or one% westward/v EDC in 80% ethanol) for 1.5 h at room temperature. Subsequently, the scaffolds were rinsed several times in Milli-Q water. The reaction was halted by treatment with ane% glycine (due west/v in water) overnight, and it was again done several times before being equilibrated in PBS for use in jail cell civilisation.
Scaffold matrices of two different shapes were used for in vitro and in vivo testing: rectangular marine collagen scaffolds for complete nasal septum replacement, with dimensions of 8×5×2 mm and disc-shaped scaffolds for in vitro iii dimensional (3D) culture, with a meridian of 3 mm and a diameter of half-dozen mm.
Scanning electron microscopy
To visualize the porosity and construction of scaffolds, scanning electron microscopy (SEM) was carried out using a Philips XL 30/ESEM with a field emission gun operated in SEM fashion. Dehydrated, freeze-stale samples were stock-still on carbon pads and sputter-coated with gold.
Stress-relaxation exam
To assess biomechanical properties, stress-relaxation tests were performed co-ordinate to Stok et al.41 Prior to biomechanical testing, samples were equilibrated for xv min in PBS at RT. Measurement of the exact scaffold height and confined compression testing were carried out in a standard materials-testing machine (Zwick, Roell) using a 5 N load cell and an indenter with a diameter of 1.38 mm. The preload was gear up to 0.one N. Samples were placed in a cylindrical confining compression chamber containing PBS. Measurements were carried out with a stress-relaxation indentation in three strain steps: 5%, xv%, and 25% of the specimen thickness. After each strain step, the specimens were left to relax for several minutes at that strain until equilibrium was reached. Throughout the testing, strength, displacement, and fourth dimension information were recorded. Subsequent analysis of the E-modulus was performed by using testXpert® software (Zwick, Roell).
Assessment of pore size
For the estimation of pore size, scaffolds were embedded in CryoMolds (Sakura) with the Tissue Tek automated embedding system (Sakura), frozen at −20°C, and cut into 40 μm slices (Leica CM 3050 Due south; Leica Biosystems). Horizontal sections (each n=3) of the middle of the scaffolds (north=3) were analyzed by lite microscopy. The boilerplate pore size is given as the hateful±standard deviation.
Isolation of main human (hCh) and rat (rCh) nasal septum chondrocytes
Human nasal septum cartilage biopsies were obtained during routine septoplasties and septorhinoplasties in the Department of Otorhinolaryngology, Academy Medical Center Ulm. Donor historic period ranged from 18 to 39 years, with an boilerplate age of 22±8 (total n=five, gender ratio female person/male ane/4). Cartilage harvesting was approved by the Ulm University Ethical Committee (No.: 152/08).
Primary rat nasal septum chondrocytes (rCh) were isolated from freshly harvested pieces of rat septum cartilage. Rat and man cartilage samples were rinsed in culture medium: DMEM/Ham's-F12 (Biochrom), supplemented with 10% FBS (Biochrom) and 0.5% gentamicin (PAA). Cartilage was minced and transferred to digestion medium: civilization medium containing collagenase type Two (0.iii%, Worthington). The cartilage was so incubated for 18 h at 37°C in a shaking water bath. Cells were pelleted by centrifugation, and total prison cell number and vitality were determined. Chondrocytes were seeded at a density of 0.v×104 cells cm2.
When cultures reached 80%–90% confluence, cells were detached by trypsinization, and so counted, and cryopreserved to ensure that chondrocytes were treated equally and that but cells in passage i were used. To avoid growth gene add-on (with a view toward later in vivo clinical utilize), rCh and hCh were amplified in civilisation medium only. All cell culture experiments were performed at 37°C and 5% CO2 under humidified weather.
In vitro evaluation of marine collagen scaffolds
Seeding and in vitro 3D culture of homo and rat chondrocytes
Prior to seeding, marine collagen scaffolds were incubated in culture medium for 24 h to arrange the pH and rehydrate the scaffold matrix. rCh and hCh were thawed and grown to fourscore%–90% confluence in monolayer culture. Cells were detached and resuspended in civilisation medium to achieve a final cell concentration of 5.0×x6 cells mL−1. V scaffolds were placed together and tightly packed in one well of a 24-well plate, and the scaffolds were seeded past adding 1 mL of cell suspension (equivalent to ane×10six cells/scaffold). To enable cell adhesion, seeded scaffolds were incubated for 1 h.
In vitro 3D culturing of hCh and rCh was carried out past using chondrocyte differentiation medium (NH Chondro Diff Medium; Miltenyi) supplemented with 0.v% gentamicin. The medium was supplemented with specific growth factors, simply the exact formulation of the medium is proprietary and non disclosed by the provider. Scaffolds seeded with rCh and hCh were analyzed on days 7, fourteen, and 21.
Cytotoxicity testing
To determine the cytotoxic effects of marine collagen scaffolds, testing was performed using rCh, hCh, and the murine fibrosarcoma prison cell line L929, as described previously, according to international standard ISO 10993-5:2009.42
Quantitative analysis for Deoxyribonucleic acid
The number of hCh growing on the marine collagen scaffold surface, and the number growing within the scaffold matrix, was estimated by performing Hoechst assay,43 equally described previously.44
Quantitative DMMB-assay for sulfated GAGs
Samples were snap-frozen and freeze-stale (Christ blastoff one–4 freeze dryer). Unseeded and seeded (vii, 14, and 21 days, each due north=6) marine collagen samples were digested overnight in a solution of 50 μg mL−1 proteinase K in 100 mM Yard2HPO4 (pH eight.0) at 56°C. Subsequent measurement of sulfated GAGs was performed, as recently published44 and described by Barbosa.45
Real-time PCR
The de novo synthesis of aggrecan (ACAN), collagen blazon I (COL1A1) and II (COL2A1), and versican (VCAN) by hCh was investigated in long-term 3D culture by analyzing mRNA expression. After 0, 7, fourteen, and 21 days (each n=6), the culture medium was removed, and the RNA of the day 0 monolayer civilisation chondrocytes was immediately isolated. Seeded scaffolds were snap-frozen and homogenized in 500 μL RLT-buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma) by using a tissue lyser (Qiagen) for 5 min at l Hz. RNA was isolated and purified using the RNeasy Mini kit according to the manufacturer'south instructions. Harvested RNA was adapted to a concentration of 50 ng μL−one and kept on ice.
I-step existent-time PCR was performed using the Real Fourth dimension Ready RNA Virus Master Kit (Roche) and Universal Probe Library (UPL, Roche). The primers that were used are shown in Tabular array 1. Reverse transcription was carried out at 58°C for 8 min, and the initial denaturation occurred at 95°C for 30 s. Amplification was performed in 2 steps for 45 cycles: 95°C for 1 south, followed past 60°C for xx s. Duplicates of each sample were performed. CP median values were adamant using a LightCycler 2.0 (Roche) and the Roche Light Cycler Software version 4.i. Relative gene expression was calculated by using the two−ΔΔCT formula.46 GAPDH was used equally a reference factor.
Table 1.
Summary of Investigated Genes, Specific Primers, Expected Fragment Size, and Used Probes
| UPL probe | Primer left | Primer right | Amplicon (nt) | |
|---|---|---|---|---|
| Target cistron | ||||
| Aggrecan (ACAN) | # 79 | v′-tgcagctgtcactgtagaaactt-3′ | 5′-atagcaggggatggtgagg-3′ | 112 |
| Collagen type I (COL1A1) | # fifteen | 5′-atgttcagctttgtggacctc-3′ | 5′-ctgtacgcaggtgattggtg-3′ | 126 |
| Collagen type Two (COL2A1) | # nineteen | 5′-ccctggtcttggtggaaac-3′ | 5′-tccttgcattactcccaactg-3′ | 88 |
| Versican (VCAN) | # 54 | five′-gcacctgtgtgccaggata-3′ | 5′-cagggattagagtgacattcatca-3′ | lxx |
| Housekeeping | ||||
| GAPDH | # sixty | 5′-gctctctgctcctcctgttc-3′ | 5′-acgaccaaatccgttgactc-3′one thousand | 115 |
In vivo evaluation of marine collagen as a cartilage replacement fabric
Seeding and 3D culture of rCh on marine collagen matrices prior to in vivo biocompatibility evaluation
3 dimensional culture of marine collagen scaffolds prior to in vivo biocompatibility evaluation was carried out by seeding scaffolds with rCh. Seeding and culture was performed every bit described in the section Assessment of pore size, except that all seeded scaffolds were transferred and cultured in culture medium to avoid the employ of growth factors. Scaffolds were precultured for one week. Deposition of GAGs subsequently 7 days was compared to unseeded scaffolds and quantified as described in section Quantitative assay for Deoxyribonucleic acid.
Creature model
An inbred strain of Lewis rats was used to minimize the risk of graft rejection due to MHC incompatibility. This approximates the autologous state of affairs. Male Lewis rats (weight: 300–350 thou) were anesthetized by the intraperitoneal injection of ketamine (0.v mg kg−1 bodyweight) and xylazine (11.5 mg kg−1 bodyweight).
Rats were intubated and fixed in a stereotactic alignment system by using nontraumatic ear bars and an incisor clamp (Small Creature Stereotaxic Instrument, David Kopf Instruments).
Later shaving and local skin disinfection, the skin was incised under sterile conditions, and the nasofrontal and nasomaxillary sutures (Fig. 1a) were exposed. After weakening these sutures with a ii mm diamond burr (PROXXON MICROMOT l/E Niersbach), a rhinotomy was performed using a 4 mm chisel. The olfactory organ was temporarily opened past moving the nasal bones anteriorly, using the stock-still pare of the snout as a hinge.
For orthotopic nasal septum replacement, the nasofrontal (*) and nasomaxillary (>) sutures were exposed and opened, whereas the internasal suture (°) was maintained (a). After detaching the mucosa, the complete nasal septal cartilage was removed (b). Color images bachelor online at www.liebertpub.com/tea
The cartilaginous septum was exposed using 2.3×magnification. The perichondrium was frankly dissected, and the complete cartilaginous septum was removed (Fig. 1b). Between the 2 mucoperichondrial flaps, the engineered scaffolds were placed in the original position of the native cartilage. Two drops of xylometazoline solution (0.025% mL−i, OTRIVEN® decongestant nose drops for infants, Novartis) were administered in the wound, the nasal bones were repositioned, and the skin was sutured using DECLENE 5-0.
After one, iv, and 12 weeks, rats were euthanized, and the whole septal area was assessed past macroscopic, histological, and immunohistochemical analyses.
Experimental groups
We established four experimental groups of animals (Table 2). The first grouping was implanted with unseeded collagen scaffolds, the second group was implanted with seeded collagen scaffolds. The third grouping was the so-called "sham-grouping", in which the nasal septal cartilage was removed and replanted inside the same surgery. For the control group, the entire nasal septal cartilage was removed without any replacement (Table two). The initial number of operated animals per experimental group was 24, to explant eight specimens after each one, 4, and 12 weeks.
Table ii.
Groups and Number of Evaluated Animals (Animalsevaluated)
| Weeks | ||||
|---|---|---|---|---|
| Grouping | 1 a | iv | 12 a | |
| Unseeded | animalsevaluated | 8 | 6 | eight |
| + | 8 | 4 | 3 | |
| − | 0 | 2 | v | |
| Seeded | animalsevaluated | viii | seven | eight |
| + | 0 | 3 | ane | |
| − | 8 | 4 | 7 | |
| Sham b | animalsevaluated | 4 | half-dozen | 8 |
| + | 0 | 0 | 0 | |
| − | 4 | 6 | 8 | |
| Command b | animalsevaluated | 8 | 8 | 7 |
| + | viii | half dozen | 5 | |
| − | 0 | two | two | |
Histological and immunohistochemical analyses
In vitro and in vivo samples were fixed in iii.5%–three.vii% neutral buffered formalin (Fischar), embedded in methane series, and sectioned at 3–5 μm. Sections were heat fixated at 56°C for 24 h. Prior to staining, the sections were deparaffinized and rehydrated.
Histological staining
Alcian blue (AB) staining combined with hematoxylin was used to visualize cell migration and cell distribution in the 3D cultured scaffolds, and this staining besides enabled detection of newly synthesized acidic sulfated proteoglycans within the explanted matrices and long-term cultured scaffolds.
Immunohistochemical detection of collagen blazon I, Two, and aggrecan
Immunohistochemical staining for collagen blazon I (anti-rat: ab34710, Abcam, man: Serotec), collagen blazon 2 (anti-rat and anti-human: II-II6B3; Developmental Studies Hybridoma Bank), and aggrecan (anti-rat: Millipore, anti-human: Serotec) was performed past using the LSAB+Arrangement-HRP (Dako) and AEC chromogen (Dako).
In brief, for detection of collagen type I, sections were digested with proteinase One thousand for five min, and later on incubated with the master antibody for 30 min at RT. Sections for detection of collagen type 2 were digested with ane% hyaluronidase (Sigma; H3506-100 MG; in PBS) and 0.2% pronase (Calbiochem, in PBS), each for 15 min at 37°C. Primary antibiotic was added for one h. For detection of aggrecan, sections were pretreated with 0.5 U mL−1 chondroitinase ABC (Sigma) in PBS for 30 min at RT and were and so incubated with primary antibody for thirty min at RT.
Histopathological evaluation of the biocompatibility index
For the determination of the in vivo biocompatibility index (bi), septal tissue and surrounding tissue were harvested en bloc. H&E stainings were evaluated according to DIN EN ISO 10993-half dozen, appendix East. Histological characteristics, such as inflammatory reactions, necrosis, fibrosis, fibroplasia, fatty infiltration, and the presence of polymorphous nuclear (PMN) cells, monocytes/macrophages, lymphocytes, plasma, and behemothic cells were determined in the half-quantitative evaluation system. Based on the histological results, a nomenclature score was estimated for each slide and explant. The integral (whole-number) classification score incorporates the presence and frequency of the different prison cell types within the implanted scaffold matrix, and in the surrounding tissue. Numbers from 0 to v were used for description (0=absent-minded, 1=slight, ii=moderate, iii=marked, and 4=severe). The total median and its respective median departure were calculated for each group and prison cell blazon or for the evaluated tissue reaction (fibrosis, fibroplasia, and fatty infiltration). binorm was defined as the difference between the total median of each time signal and the full median of the sham group. To exclude any exam bias, the microscopic assay and the calculation of bi were conducted by using CellMed AG in a double-blind study.
Statistical analysis
The Wilcoxon–Isle of man–Whitney test was used to evaluate the significance of the in vitro cytotoxicity and GAG degradation prior to in vivo application (α=0.05). Considering the normality test failed, the Kruskal–Wallis one-way analysis of variance on ranks was used for the evaluation of significance (level of significance α=0.05) for jail cell number, cistron expression analysis, GAG deposition, and bi (α=0.05). The relative per centum of jail cell vitality (cytotoxicity testing), the cell number, the relative percentage of GAG content, evaluation of bi, and the gene expression are reported as median±median deviation (MD).
Macroscopic results were statistically analyzed with a multiple logistic regression. Grouping and time were determining factors, whereas the occurrence of a macroscopically visible septal perforation (yeah/no) was the dependent variable. The results were adjusted for the furnishings of other determining factors in the model to provide "odds ratios" (OR), including p-values.
Results
Pore size and East-modulus of marine collagen
SEM assay of unseeded scaffolds revealed a high porosity and a homogenous pore distribution, with distinct pore interconnectivity visible in the horizontal (Fig. 2a) and vertical cross sections (Fig. 2b). All empty pores in the marine collagen scaffolds had a diameter in the range of 40–200 μm. In horizontal sections of the scaffold centre, the pore diameter was 74±18.1 μm. In superficial zones, the pore diameter was 50±xx μm. The E-modulus was betwixt xv and 25 kPa.
SEM analysis of marine collagen scaffolds reveals high porosity and homogenous pore distribution on the scaffold surface (a) and within the consummate scaffold matrix (b). Both views, the superficial superlative view (a) and the vertical cross department, demonstrate the distinct interconnectivity of the scaffold matrix.
Marine collagen is not cytotoxic
The cultivation of L929, rCh and hCh in marine collagen extracts revealed no cytotoxic effects (Fig. 3). Viability for these cell types was 99.65%±1.18%, 103.75%±1.82%, and 98.24%±1.49%, respectively. Cell viabilities between 70% and 100% showed that there were no cytotoxic components in the matrix extracts. The viability of L929 (31.70%±five.62%), rCh (26.81%±four.88%), and hCh significantly decreased when incubated in DMSO (18.21%±2.98%). Cell viabilities between 0% and 40% reverberate stiff cytotoxic effects. The number of initially seeded cells per each well slightly varied due to technical reasons. Therefore, slightly higher cell numbers cause, as a byproduct, values college than 100%.
In vitro cytotoxicity test of marine collagen. L929, rCh, and hCh were used as indicator cells. All cells cultured in negative command (dotted line, 100%), or in undiluted sample extracts demonstrate a loftier viability and reflect the noncytotoxic upshot of the extracted marine collagen. No significant differences between negative controls and extracts were detectable. Compared to these results, the cytotoxic event of 10% DMSO, used as positive control, is meaning (each *p<0.05).
Quantification of homo chondrocyte number and GAG production in marine collagen scaffolds
Afterward seeding, the number of adherent hCh was one.21×105±2.56×teniv. After 7 (iv.64×xfive±9.07×x4), 14 (five.22×10five±3.77×104), and 21 days (7.23×10v±2.09×105), the cell counts had increased significantly, as determined by the Hoechst assay (Fig. 4a).
Changes in cell number (a) and content of sGAG (b) on marine collagen scaffolds after 0, 7, 14, and 21 days of iii dimensional (3D) civilisation with hCh. Marine collagen scaffolds were initially seeded with 1×10half-dozen hCh. During 3D culture, the cell number increased significantly. GAG neo synthesis per mg dry out weight of unseeded compared to seeded and cultivated scaffolds. GAG synthesis significantly increased during the cultivation menses of 21 days, due to enhanced GAG accumulation by human chondrocytes (*p<0.05). sGAG, sulfated glycosaminoglycan
Effigy 4b shows the significant increase in GAG content (DMMB-analysis) during the start week of 3D culture. A GAG content of three.3±ii.35 μg mg−one (per mg dry weight) was measured in native marine collagen scaffolds. After seven days in civilisation, the GAG content significantly increased to 19.vii±1.9 μg mg−1. Afterward 14 (20.6±2.03 μg mg−1) and 21 days (18.97±2.63 μg mg−one), a slight merely not meaning increase in GAG content was observed.
Human being chondrocytes redifferentiate on marine collagen scaffolds and produce cartilage-specific ECM
Following distension in monolayer culture, gene expression of all examined ECM markers (ACAN, COL1A1, COL2A1, and VCAN) was downregulated and detectable only at very depression levels. The values of ACAN, COL1A1, COL2A1, and VCAN expression (0 day) were set to 1 (Fig. 5a, b, dashed threshold line).
Relative gene expression of chondrogenic, cartilage-specific marker genes (ACAN and COL2A1) (a), and marking for dedifferentiation (COL1A1 and VCAN) (b), in 3D culture for 0, 7, 14, and 21 days using hCh. Relative gene expression was calculated by means of the 2-ΔΔCT formula. GAPDH was used as reference cistron and values for COL2A1, ACAN, COL1A1, and VCAN expression on day 0 (monolayer culture) were set to 1 (dashed threshold line). COL2A1 and ACAN (a) were expressed at significantly college levels compared with twenty-four hour period 0 (each *p<0.05). With proceeding civilization fourth dimension a meaning increase in cistron expression of ACAN was detected ( + p<0.05) while expression of COL2A1 remained on a stable level. Compared to the bench marker of day 0, VCAN and COL1A1 (b) expression increased significantly (each *p<0.05) during the start 14 days of cultivation. Afterwards, gene expression of both dedifferentiation markers significantly decreased during 3D civilization (°COL1A1 p<0.05; # VCAN p<0.05).
After xiv days, ACAN expression increased further (ACAN 38.25±12.99-fold; each p<0.05) while COL2A1 expression slightly decreased in comparing to 24-hour interval 7, although still existence college than after monolayer culture (COL2A1 14.88±x.lx-fold). Between xiv and 21 days, no pregnant change in factor expression occurred for COL2A1 and ACAN, and a stable expression pattern was detected (Col2A1 13.17±18.77-fold; ACAN 35.17±17.01-fold).
During the commencement 7 days of 3D culture, factor expression of COL1A1 (418.thirty±180.lxx-fold) and VCAN (86.53±13.93-fold), generally accustomed every bit markers of chondrocyte dedifferentiation,47 significantly increased compared to day 0 (Fig. 5b). After xiv days, COL1A1 and VCAN expression increased farther (COL1A1 574.13±134.14-fold; VCAN 122.34±14.59-fold; each p<0.05). However, after 21 days, gene expression of both of these dedifferentiation markers significantly decreased (COL1A1 229.96±43.37-fold; VCAN 53.71±12.93-fold; each p<0.05).
These results were confirmed by histological and immunohistochemical staining (Fig. 6), and past GAG quantification (Fig. 4b). AB staining (Fig. 6a–c) and specific immunohistochemical staining for aggrecan (Fig. 6d–f) in marine collagen scaffolds seeded with hCh and rCh (information non shown) cultured for up to 21 days allowed the visualization of the enhanced GAG deposition in 3D culture. GAG and aggrecan accumulation was detected by increased staining, starting within the scaffold's periphery where chondrocytes had adhered after seeding. With progressing chondrocyte migration and proliferation, GAG and aggrecan accumulation spread within the scaffold matrix (Fig. 6a–c, d–f).
Histological AB (a–c) and immunohistochemical staining for detection of ECM neo synthesis (Agg, collagen type I, and 2; d–l) in marine collagen scaffolds seeded with hCh starting from twenty-four hour period vii (a, d, g, j) until solar day xiv (b, e, h, m) and 21 (c, f, i, l). The AB (a–c) staining reflects enhanced GAG deposition during 3D culture. 7 days later seeding (a) GAG accumulation was detectable and increased until solar day 14 (b) and 21 (c). A visible increase of GAG and aggrecan accumulation from day 7 (d) to day 14 (e) and 21 (f) was demonstrated. Neo synthesis of collagen type II (1000–i) started afterward the first calendar week and visibly increased during further civilization after 14 (h) and 21 days (i). Collagen type I was nowadays within the whole scaffold starting inside i week after initial seeding (j) and increasing to day fourteen (k). Distribution of collagen blazon I proceeded into the eye of the scaffolds later on 21 days. Nevertheless, the intensity of the staining remained at a comparable level (l). *periphery of scaffold; °center of scaffold;→scaffold fibers; 
cell nuclei. Color images available online at world wide web.liebertpub.com/tea
The chapters of hCh (Fig. 6) and rCh (data not shown) to synthesize ECM products was additionally examined past immunohistochemical staining for collagen type I (Fig. 6j–l) and collagen blazon II (Fig. 6g–i). Production of collagen type I by hCh was detectable on days 7 (Fig. 6j), 14 (Fig. 6k) and 21 (Fig. 6l). No collagen blazon Ii synthesis was detected subsequently 7 days. After 14 and 21 days, collagen type Two synthesis increased and progressed to the center of the scaffold as jail cell migration occurred (Fig. 6g–i). rCh displayed a similar design of zipper and ECM neo synthesis (data not shown). The distribution of cell nuclei showed that hCh and rCh initially adhered to the scaffold surface and outer pores of marine collagen. Subsequently, within the first week, both types of chondrocytes migrated to form homogenous cell distributions after 14 and 21 days.
Three dimensional culture of rCh on marine collagen scaffolds prior to in vivo application enables synthesis of cartilage-specific matrix proteins
RCh became attached to the scaffold surface. Within 7 days, they migrated throughout the entire scaffold. Aggrecan synthesis increased with culture time from day 1 (Fig. 7a) to day seven (Fig. 7d). Synthesis of collagen type I (Fig. 7b, e) and type II (Fig. 7c, f) both increased over the culture period. GAG content significantly increased later 7 days (Fig. 8) (23.54±ii.14 μg mg−ane) compared with unseeded scaffolds (2.81±1.82 μg mg−1).
Marine collagen scaffolds seeded with rCh. While later one 24-hour interval (a) aggrecan was not detected, aggrecan deposition (blood-red) became visible afterward vii days (d). Collagen type I synthesis visibly increased (red) from day 1 (b) to day 7 (east). Within the tightly seeded marine collagen scaffold slight accumulation of collagen blazon 2 (brown) (c,f) was detectable subsequently 7 days (f).→scaffold fibers; *seeded surface. Color images available online at www.liebertpub.com/tea
Presence of GAG in unseeded marine collagen and in scaffolds seeded with rCh. GAG content significantly increased during the tillage flow of 7 days (*p<0.05).
Orthotopic fauna model for nasal septum replacement, and the frequency of septal perforations
The number of septal perforations was significantly different betwixt the experimental groups. Fewer perforations were detectable inside the replacement groups (seeded and unseeded scaffolds) than in the animals of the control group. Specifically, when seeded scaffolds were implanted, none of eight animals had a septal perforation after 1 week. After 4 weeks, three out of vii animals had a perforation, and afterward 12 weeks, i in eight of the animals had a detectable perforation (Table 2). In animals transplanted with unseeded marine collagen matrices, we found perforations in all eight individuals afterward one week, in four of six animals afterward 4 weeks and in three of eight animals afterwards 12 weeks. In the command group, removal of the septal cartilage without replacement resulted in eight of eight animals having a septal perforation after one week, half dozen of viii animals having a perforation after 4 weeks and five of seven animals having a perforation after 12 weeks. To evidence that the surgical procedure itself does non affect the results, a sham group was established. Following all 3 explantation time points, no septal perforations were detectable. Furthermore, at all time points, the number of septal perforations was significantly (p<0.05) higher in the control grouping compared to seeded scaffolds. Residual scaffold cloth was detected in merely a few animals. Afterward 1 week, marine collagen remnants were detected by HE staining in the unseeded (v/8) and seeded group (5/10). AB staining revealed a low level of sGAG synthesis in some of the unseeded scaffolds (Fig. 9). Collagen blazon I and 2 were detected in small amounts (information not shown). Furthermore, remnants of marine collagen were detected in two animals of the unseeded grouping after 4 weeks and in one animal afterward 12 weeks. Due to the death of ten% of the operated animals, the number of evaluated animals was sometimes lower than 8 animals.
AB staining of remaining scaffold textile (*) of unseeded marine collagen scaffolds after one week in vivo. Unseeded collagen scaffolds, demonstrate a slight accumulation of GAGs (#). Color images available online at www.liebertpub.com/tea
Evaluation of the biocompatibility of marine collagen
Changes in total and differential leukocyte frequency with progressing implantation time indicate the presence of acute and chronic inflammatory reactions. PMNs prove the astute inflammatory response at shorter times (1 week), whereas increased occurrence of macrophages and lymphocytes indicate persisting chronic inflammatory reactions within the vicinity of implants. PMNs were rarely detected (nearly absent-minded) in all groups and time points. The highest level of PMN infiltration occurred after 1 calendar week in unseeded and seeded scaffolds, and this level decreased until week 12. No significant differences in the median scores of PMNs were institute between any of the experimental groups (Table three).
Table 3.
Summarized Nomenclature Scores of Seeded and Unseeded Scaffolds one, iv, and 12 Weeks Afterwards Implantation Compared to Sham Group
| Grouping | Weeks | Polymorph- nuclear cells | Lymphocytes | Plasma cells | Macrophages | Giant cells | Necrosis | Fibroplasia | Fibrosis | Fatty infiltrate | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Unseeded | 1 | Median | 0.00 | one.00 a | 0.00 | 1.00 a | 0.00 | 1.50 a,b | 1.00 | one.l a | 0.00 |
| ±MD | 0.22 | 0.47 | 0.00 | 0.22 | 0.22 | 0.75 | 0.00 | 0.50 | 0.00 | ||
| iv | Median | 0.00 | 1.00 a | 0.00 | one.00 a | 0.00 | 0.l | ane.00 a | 1.50 a | 0.00 | |
| ±Doctor | 0.28 | 0.28 | 0.00 | 0.28 | 0.00 | 0.50 | 0.44 | 0.50 | 0.00 | ||
| 12 | Median | 0.00 | 1.00 a | 0.00 | 1.00 a | 0.00 | 0.50 | 1.00 a | 1.50 a | 0.00 | |
| ±MD | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.50 | 0.00 | 0.l | 0.00 | ||
| Seeded | 1 | Median | 0.00 | 1.00 a | 0.00 | ane.00 a | 0.00 | 1.00 a,c | one.00 | 2.00 a | 0.00 |
| ±MD | 0.59 | 0.44 | 0.00 | 0.20 | 0.20 | 0.79 | 0.xx | 0.35 | 0.00 | ||
| 4 | Median | 0.00 | 1.00 a | 0.00 | 1.00 a | 0.00 | 0.00 | 1.00 | ii.00 a | 0.00 | |
| ±MD | 0.28 | 0.00 | 0.00 | 0.28 | 0.28 | 0.44 | 0.00 | 0.44 | 0.00 | ||
| 12 | Median | 0.00 | 1.00 a | 0.00 | one.00 a | 0.00 | one.00 a,c | ane.00 | 1.00 | 0.00 | |
| ±MD | 0.22 | 0.38 | 0.00 | 0.22 | 0.00 | 0.22 | 0.22 | 0.47 | 0.00 | ||
| Sham | one | Median | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.00 | 1.00 | 0.00 |
| ±Doc | 0.38 | 0.38 | 0.00 | 0.38 | 0.38 | 0.00 | 0.38 | 0.00 | 0.00 | ||
| 4 | Median | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.00 | 0.00 | |
| ±MD | 0.00 | 0.00 | 0.00 | 0.24 | 0.00 | 0.41 | 0.41 | 0.00 | 0.00 | ||
| 12 | Median | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.00 | 0.00 | |
| ±MD | 0.00 | 0.41 | 0.00 | 0.00 | 0.00 | 0.00 | 0.41 | 0.00 | 0.00 |
In all groups, even in the sham group, slight infiltration of lymphocytes and macrophages was detected. Macrophages and lymphocytes were the dominant cell types in seeded and unseeded scaffolds (Table 3). However, macrophages and lymphocytes were rarely detectable in the sham group. Subsequently one, 4, and 12 weeks, the slight aggregating of macrophages and lymphocytes was significantly higher in unseeded and seeded scaffolds compared to the native cartilage of the sham group (p<0.5) (Tabular array iii). Nosotros were not able to detect plasma cells. Giant cells were as well very rarely detected. Slight necrosis was observed in seeded and unseeded scaffolds. In unseeded scaffolds, the median necrosis score was (1.l±0.75) after ane week, and this score significantly decreased later four (0.5±0.v) and 12 (0.5±0.five) weeks (each p<0.5). Further, the median necrosis score in 1 (1.0±0.79) and 12 (i.0±0.22) week seeded scaffolds was significantly higher (p<0.05) compared with the 1 (0.0±0.00) and 12 week sham group (0.0±0.24, each p<0.five).
In all experimental animals, low levels of fibroplasia and fibrosis were detectable. One and 4 weeks after implantation, fibrosis in seeded scaffolds (i calendar week: 2.0±0.35; four weeks: 2.00±0.44) was significantly higher than in the sham grouping (1 week: 1.00±0; four weeks: ane.00±0.00; each p<0.5). After four (ane.00±0.44) and 12 (1.00±0.00) weeks, fibrosis in unseeded scaffolds was significantly higher than in the sham groups (four weeks: 0.00±0.41; 12 weeks: 0.00±0.41; each p>0.5). Fatty infiltrations were not found in any of the specimens. On the basis of tissue and cellular responses, we determined the bi values (Fig. 10a) of the unseeded and seeded scaffolds, and nosotros determined the impact of the surgery itself (sham group). Ane calendar week after implantation, the bi of the unseeded scaffolds (12±i.72) was significantly higher than the bi of the sham group (three±1.75, p<0.v), indicating a moderately irritating effect. Until week 12, the bi of the unseeded scaffolds significantly decreased to a slightly irritating effect (seven.5±0.83, p<0.5). Seeded scaffolds (nine±3.16) had a lower (n.s.) bi and only slightly irritating effects after ane week when compared to unseeded scaffolds (12±ane.72). The effects of the surgical procedure itself (bi of sham grouping) significantly decreased during the first 4 weeks (1 calendar week: 3±1.75; 4 weeks: 1±0.98, p<0.five). After 4 and 12 weeks, the bi of unseeded (4 weeks: viii.5±1.17; 12 weeks: 7.v±0.83, p<0.five) and seeded (4 weeks: 7±1.56; 12 weeks: 8.5±1.00) scaffolds was significantly college than the bi of the sham group (4 weeks: i±0.98; 12 weeks: 1±1.04, p<0.5).
bi and binorm of seeded and unseeded scaffolds subsequently ane, four, and 12 weeks as median value±median deviation compared to sham grouping ( bi, a) and afterward subtraction of the bi of the sham group ( binorm , b) (*p<0.05). All detected bi values are within the slightly irritating range (Classification of bi: not irritating 0.0–2.nine, slightly irritating iii.0–8.ix, moderately irritating 9.0–fifteen.0, and highly irritating >fifteen.one).
To separately assess the influence of the seeded and unseeded matrices, the bi values were normalized through subtraction of the bi evoked by the surgical procedure (sham group). Inside 12 weeks, the binorm (Fig. 10b) decreased to 6.v (unseeded) and 7.five (seeded), reflecting the slightly irritating outcome of the implants. In that location were no differences between the groups and time points.
Discussion
Cartilage lacks an intrinsic regeneration capacity,48 which makes cartilage reconstruction challenging. Cartilage reconstruction has get a major focus of tissue applied science research.nineteen,49 For the first fourth dimension, this written report evaluated the utility of marine collagen as a scaffold for cartilage replacement. We demonstrated that in full general, marine collagen scaffolds seem to exist suitable cell carriers for clinical cartilage tissue engineering. Farther, we established a new immunocompetent rat model for the assay of tissue-engineered nasal cartilage.
We used triple helical, homotrimer collagen extracted by lyophilization of the jellyfish species Rhopilema esculentum. 30 To enhance the mechanical forcefulness, epoxy carbodiimide cross-linking was applied. Various cross-linking reagents, such as formaldehyde, glutaraldehyde, epoxy compounds, carbodiimide (EDC), proanthocyanidin and dimethylsuberimidate, have been used for the fixation and cross-linking of collagen to increase its strength and resistance to enzymatic digestion.22,50,51 However, all cross-linking agents showroom certain disadvantages, such as toxicity, instability, and poor control over the rate of cross-linking.l Cytotoxicity testing results correlate with curt-term implantation studies as Kotzar and coworkers accept stated,52 and the toxicological effects of the marine matrices were kickoff examined in vitro. No cytotoxic effects due to material components, EDC cross-linking agents or the production process were detectable, confirming the results presented previously past Song et al.22
Cross-sections of marine collagen scaffolds revealed high porosity of the scaffold matrices, with distinct interconnectivity, as has been demonstrated previously for other matrices based on jellyfish collagen.22 The pore size and architecture of biomedical materials are known to influence the diffusivity of nutrients, oxygen and serum in the matrix, and consequently, to control the nutrition of cells.22,44,53 Furthermore tissue formation54 and the rate and depth of cellular in growth in vivo are likewise influenced.55 The pore size of the examined marine collagen scaffolds cannot be rigorously controlled during the freezing process, and thus, varied quite significantly. Equally estimated by light microscopy, pore size was betwixt twoscore and 200 μm, with a main range of 74±18.1 μm in the scaffold center, and 50±xx μm in superficial zones. The literature on optimal pore size for chondrogenesis is quite heterogenous. Some authors favor small pore sizes between xv–fifty μm56 and state that in smaller pore sizes, chondrocytes are packed more than closely, and the resulting cell–cell interactions play a primal role in phenotype expression.57 Information technology has been suggested that smaller pore sizes enhance the formation of the ECM and neo cartilage.58 In contrast, Oh and co-workers59 state that larger pore sizes (between 380 and 405 μm) are optimal for chondrocytes. These differences are near likely acquired by unlike study designs with respect to cell number and the material used. Other parameters of scaffold compages design, such as pore shape, porosity, and pore interconnectivity, additionally influence cellular differentiation.60 In this report, rat and human chondrocytes were detectable on the scaffold surfaces and in the centers of the scaffolds afterwards vii days. This indicates that despite considerable variability, pore size and pore interconnectivity allowed rapid migration through the scaffold. Therefore, marine collagen scaffolds provide suitable microenvironmental atmospheric condition for the homogeneous production of cartilaginous ECM. Rapid GAG synthesis and increased mRNA expression of the cartilage-specific markers collagen blazon Ii (gene Col2A1) and aggrecan (Agg) additionally underline this finding.
In add-on to pore size, the type of collagen chosen as the implant matrix influences the morphology and phenotype of the chondrocytes. In collagen type I matrices, the expression of phenotype and biosynthetic activity has been shown to be dependent on pore diameter. Most chondrocytes cultured on such matrices retain a fibroblast-like morphology and brandish an increased proliferation rate with a concomitantly depression GAG biosynthetic activeness.57,61 It has been suggested that type II collagen scaffolds provide a better surroundings for hyaline chondrocytes than collagen type I scaffolds.61,62 Blazon 2 collagen scaffolds facilitate the maintenance of a differentiated chondrocytic phenotype. In collagen type Ii sponges, bovine and canine chondrocytes accept been demonstrated to re-limited their typical spherical shape accompanied by the production of cartilage-specific GAG and collagen type II.61,62 Comparable effects have been demonstrated for rat and man nasal septal chondrocytes in marine collagen matrices within the nowadays study. Both chondrocyte types synthesized GAG and collagen type II in the scaffold, which consists of a homotrimer collagen type similar to collagen type 2. Marine collagen possesses only one blazon of blastoff chain and a similar degree of glycosylation compared to vertebrate collagen type 2 (unpublished results). Although significant product of collagen type I was visualized until 24-hour interval 21 in vitro, gene expression analysis demonstrated a shift from collagen type I to collagen type II expression later 21 days. This indicates that chondrogenic redifferentiation with respect to collagen synthesis takes at least 21 days on marine collagen scaffolds. In contrast, aggrecan expression and synthesis is induced already after seven days in 3D civilisation.
The in vitro results led usa to the hypothesis that marine collagen is a suitable material for cartilage tissue engineering. We developed a new model of nasal cartilage repair to evaluate the material for this specific application. To the all-time of our knowledge, no rat model for nasal cartilage replacement has previously been described. Additionally, no studies are available that employ tissue-engineered cartilage in the nasal septum. Tissue replacement in the olfactory organ is complicated by the specific immunological environment, which is potentially able to modify material backdrop63 and degradation characteristics. Therefore, a subcutaneous creature model would not run across the requirements of nasal cartilage reconstruction.63–65 In our study, removal of nasal septal cartilage and reconstruction of that cartilage were carried out in the same process. No incisions or flaps of the mucosa were necessary. Thus, the model is comparable to the clinical state of affairs of septum reconstruction, in which septal correction and reconstruction is performed in one step.
Tissue and cellular host responses to local injuries include inflammation, wound healing, and foreign body responses. The host response is initially elicited by the surgical procedure itself.66,67 For biomedical applications, information technology is essential to precisely quantify the morphological alterations of cells migrating into the implant matrix68 and to evaluate the local tissue responses, such as fibrosis or fibroplasia.52,69 Chronological sequences and pathophysiological tissue responses are used to measure out host reactions to implant materials.67
During the initial implantation stage, PMNs, leucocytes, and macrophages were detectable in all engineered constructs at low levels. During the chronic inflammatory response, a decreasing level of PMNs was detected. A slight infiltration of macrophages and lymphocytes was detectable throughout the entire experiment, indicating a slightly irritating issue on the marine collagen scaffold. Equally with many other biocompatible materials,66 the inflammatory response to our implants revealed low levels of macrophages, which fused to form foreign body giant cells, after i (unseeded and seeded) and 4 weeks (seeded). Granulation tissue development, strange body reaction, and fibrosis (gristly capsule) vary in duration, depending on degradation rate.66 Due to the high in vivo biodegradability of marine collagen,22 rapid phagocytosis of marine collagen by macrophages and strange body giant cells took place within the start 4 weeks after implantation. The cellular infiltration scores and bi classification of marine collagen were low, indicating an overall minor irritating effect and slight tissue and cellular inflammation responses.
The prevention of septal perforations is an important issue in clinical septal reconstruction. Afterwards ane week, septal perforation occurred in 100% of the animals when the nasal septum was not replaced (control grouping), and it occurred in more seventy% of the animals of the control grouping after 12 weeks. These information are comparable to the clinical situation, in which perforations occur frequently when septal cartilage is non replaced.
In contrast to the results of Kaiser et al.,70 no regenerative areas were found in our model in samples of the control grouping. In their study, the authors used a rabbit model and studied cartilaginous regeneration later on 7 months. Thus, the dissimilar written report design can explicate the differences in results. To exclude septal perforations that occurred due to surgical technique, the sham group was established, and no perforations were detectable.
Although, there was no pregnant difference betwixt the groups implanted with seeded and unseeded scaffolds after 4 and 12 weeks, we detected significantly fewer perforations when the nasal septum was replaced by seeded scaffolds in comparison to the control group. This indicates the positive effect of seeded scaffolds for the effective prevention of septal perforations. This is emphasized past the significant difference betwixt seeded and unseeded scaffolds after 1 week. Our results suggest that septal replacement by seeded marine collagen matrices is more effective considering less perforations were detectable with seeded matrices. Therefore, seeding with chondrocytes is an important gene in nasal cartilage engineering, at least under the experimental conditions used in this model. Overall, the in vivo results indicate that marine collagen is a promising new scaffold for nasal cartilage tissue technology.
Time seems to exist an additional factor influencing septal perforations, as confirmed by the observation that afterward 12 weeks, fewer perforations were detectable than subsequently 1 week in the unseeded scaffold group. Spontaneous regeneration, as demonstrated by Kaiser et al.70 in their rabbit model after 7 months, was non detected histologically but might have reduced the number of septal perforations. This is supported by the tests of unseeded marine collagen scaffolds.
The rapid biodegradability of marine collagen has been demonstrated before22 and was confirmed by our information. The relatively low biomechanical stiffness of the textile22 necessitated conscientious treatment during surgery merely did not disturb the biological function of the implants. More complete cantankerous-linking of the marine collagen could be helpful from a surgical point of view.
In summary, our findings indicate that marine collagen is a safe, natural collagen matrix without cytotoxic furnishings. At the aforementioned fourth dimension, marine collagen offers excellent biocompatibility, with only slight prove of local inflammatory reactions. Scaffolds are suitable for effectively preventing nasal septal perforations, particularly when seeded with autologous chondrocytes. Thus, marine collagen is a promising candidate for cartilage tissue engineering. The newly established rat model can exist used to compare the backdrop of diverse biomaterials for orthotopic nasal cartilage repair.
Acknowledgments
The collagen type II antibody (Two-II6B3; isotype mouse IgG1) was obtained from the Developmental Studies Hybridoma Depository financial institution, developed under the auspices of the NICHD and maintained by the Academy of Iowa, Section of Biological Sciences, Iowa Metropolis, IA 52242.
The authors acknowledge the excellent technical assistance of M. Jerg, K. Urlbauer, G. Cudek, and V. Fuss (CellMed AG, Alzenau, Germany). Special thanks to Birgit Hoyer (Carl Gustav Carus University Medical Center and Medical Department of the Technical Academy Dresden, Germany) for providing the SEM images.
The work was supported past a grant from the European Committee (EXPERTISSUES, Contract No. 500283) and by CRM (Coastal Inquiry & Direction, Kiel, Germany).
Disclosure Statement
No competing fiscal interests exist.
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Can Collagen Two Help Repair Sinus Cartlidge,
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