Impact of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum bones after sternotomy

Median sternotomy is the surgical method of choice for many procedures where one of the main problems is the long post-operative wound healing process leading to sternal dehiscence and the development of infection. This leads to prolonged hospital stay and increased mortality due to post-operative complications. A promising solution seems to be the use of allogeneic chondrocytes for wound treatment, whose properties in the field of cartilage reconstruction are widely used in medicine, mainly in orthopedics. In the present study, we investigated the effect of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum after sternotomy. We optimized the culture conditions for the isolated chondrocytes, which were then applied to the sternal incision wound. Chondrocytes in the culture were assessed on the basis of the presence of chondrocyte-specific genes: Sox9, Aggrecan and Collagen II. In turn, the histopathological and immunohistochemical evaluation was used to assess the safety of implantation. In our work, we demonstrated the possibility of obtaining a viable culture of chondrocytes, which were successfully introduced into the sternal wound after sternotomy. Importantly, implantation of allogeneic chondrocytes showed no significant side effects. The obtained results open new possibilities for research on the use of allogeneic chondrocytes in the process of accelerating wound healing after median sternotomy.

The causes of delayed sternum healing are: lack of primary synostosis, poor wound healing or premature mobilization.In turn, mortality due to sternal instability used to reach 50%, but medical progress and the development of new technologies have contributed to a significant reduction to about 10%, which is still a serious problem 15,16 .In addition, the development of these complications is influenced by many risk factors associated with the patient's comorbidities, such as obesity, osteoporosis, heart failure, as well as his lifestyle and the surgery performed 4,14,17 .
Various methods are used to accelerate the healing of sternum wounds, reducing the risk of post-operative complications.These methods are constantly being improved 15,18,19 .Among them, there are innovative techniques involving the use of specially prepared ribs and a scapula to function like a previously removed sternum 20,21 .Despite the promising results of these methods, they are still very invasive and carry a high risk of complications and mortality.Sternum dehiscence remains a significant problem and there are no effective methods to prevent it 15,19 .In the face of these issues, it seems clear that the problem affects a wide and diverse population.
The solution to this problem could be the use of allogeneic chondrocyte cultures taken from the sternum cartilage.The chondrocyte culture technique is widely used in orthopedics for the treatment of knee defects, where autologous chondrocytes are obtained directly from the injured patient's knee joint [22][23][24][25] .This technique produces impressive results.After six months, the lesion is not visible in endoscopic control, it is completely overgrown and covered with cartilage 23 .
Herein, we have proved that it is possible to obtain a viable culture of chondrocytes, that have been successfully introduced into the sternal wound after sternotomy without causing side effects.

Swine chondrocytes demonstrate expression of specific markers
Swine chondrocytes were successfully isolated from domestic pigs (Sus scrofa domesticus) xiphoid cartilage.The cells were cultured in 4 different media serum in order to select the one in which the cell proliferation was the highest.After the adhesion time, swine chondrocytes acquired a specific phenotype (Fig. 1).
We then confirmed the phenotype of the obtained porcine chondrocytes by immunocytochemical (ICC) analysis.Our results revealed that the established cells were positive for specific chondrogenic markers: Sox9, Collagen II and Aggrecan (Fig. 2).
In order to further assess the quality of swine chondrocytes, we checked the presence of Sox9, Collagen II and Aggrecan in the analysed cells.We have proven that chondrocytes cultured in all tested media serum reveal the

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To use chondrocytes with the highest proliferation rate, we evaluated the proliferative capacity of swine chondrocytes cultured in 4 different serum media.For this purpose, we performed the MTT assay at 4 time points (Fig. 4).The results showed that chondrocytes cultured in commercial and self-prepared pig serum had the highest survival ratio.Considering this, we selected this serum for further analysis and in vivo studies.

Swine chondrocytes display expression of specific chondrogenic genes
To confirm the functionality of the obtained cells, we performed an analysis of chondrocyte-specific gene expression.Our previous analyzes showed that the highest level of proliferation was obtained when chondrocytes were cultured in the commercial and self-prepared pig serum.Therefore, the relative gene expression of chondrocytes cultured in two different serum media and at different passages was compared (Fig. 5).
We observed the highest gene expression for commercial and self-prepared pig serum at passage 0 in all analysed genes.Sox9 expression was higher in all tested passages (0-3) compared to Aggrecan and Collagen II, where we observe a decrease in gene expression already at passage 1.

Morphological and biochemical analysis of blood confirms the safety of allogeneic chondrocyte implantation
To assess the safety of allogeneic chondrocyte implantation, we analyse blood samples taken before the procedure, 3 and 6 weeks after the procedure in both, the control and study groups.Administration of a new medical glue to enhance the healing process of bone structures did not induce any measurable septic reaction in serum samples in the study group.Results from TNF, IL-1 and IL-6 taken at baseline, and at 3 and 6 weeks of follow up showed no statistically significant variation and were comparable between time points and groups (Table 1).CRP levels were statistically significant after 3 weeks of observation (p = 0.01) with lower values in the control group compared to the study group.After 6 weeks of observation, this difference was no longer noticeable (p > 0.05).CBC and biochemistry were normal in both groups with some minor deviations from reference ranges with no clinical relevance compared to physical examinations of the animals (Supplementary Materials; Table S1).Although some animals developed abscesses at necropsy (Supplementary Material: Table S2), no blood results suggested generalized septicemia and localized infections.In conclusion, the obtained results indicate that the use of medical glue did not cause any adverse effects in animals that completed the observation period after 3 and 6 weeks.

Immunohistochemical evaluation of Aggrecan and Collagen II expression
We observed the expression of Aggrecan and Collagen II in the extracellular matrix and cytoplasm of chondrocytes in the growing cartilage in all examined sections of the sternum (Fig. 6).
There were no significant differences between the control and the study group after 3 and 6 weeks from the implantation of medical glue containing allogeneic chondrocytes into the sternum wound after medial sternotomy.We also did not observe differences in cellular and tissue expression of analyzed proteins between different parts (cranial, medial, caudal) of the sternum.

Implantation of medical glue with allogeneic chondrocytes does not interfere with the healing process of the sternum wound after medial sternotomy
Histopathological examination performed 3 and 6 weeks after the procedure, both in the control group and the study group in terms of the assessment of basic inflammatory parameters and the presence of cellular structures, did not show any significant differences (Table 2).We observed similar histopathological features of healing in both groups.No cases of cartilage neoplasia were observed, although cases of impaired maturation were seen in both groups-presence of polymorphic chondrocytes and blurred stratification and polarization of chondrocyte arrangement.These lesions coexisted at the periphery of the mature cartilage, so they should be considered as leading to mature cartilage (Figs. 7, 8).

Discussion
Wound healing of the sternum after median sternotomy, currently the most common method of surgical access to the internal thoracic organs, remains one of the most significant post-operative complications 26,27 , which affects the length of hospital stay and patient mortality 28 .One of the key elements is to achieve rapid synthesis of the sternum that provides adequate stabilization and restores the continuity of its structure, thus reducing the possibility of developing a deep wound infection [6][7][8]29 .
This seems difficult to achieve due to the impact of the patient's risk factors, i.e. obesity, age, gender, which impede the proper course of the healing process 30 .It has been shown in literature that the use of existing reconstructive techniques reduces the length of hospital stay, but does not reduce the risk of infection 31 .Furthermore, the use of chondrocyte cells suggests that it may be very beneficial in patients with osteoporosis, in whom the regeneration process is impaired 32 and in patients with a paramedial sternotomy wound requiring rapid and effective stabilization 33 .A promising solution could be the use of cell cultures, in particular chondrocytes.Under the right conditions, these cultures are successfully used in the treatment of cartilage defects in knee joints and osteoarticular lesions 34,35 .The extraction of chondrocyte cells from articular cartilage, auricle or trachea for the regeneration of cartilage defects is an increasingly used technique [36][37][38] .It has been shown in the literature that it is possible to obtain viable chondrocytes using a digestion medium based on collagenase II at concentrations in the range of 0.1-0.2% 38, which was confirmed in our study (Fig. 1).In addition, significant differences in the level of cell proliferation were observed depending on the culture medium used 39 .This level was highest for the commercial and self-prepared pig serum compared to the commonly used FCS and FBS serum (Fig. 4).Clear differences in cell phenotype were also observed.This shows what effect the use of specific cell medium has on the yield of the obtained culture.
Chondrocyte cultures are characterized by the expression of characteristic genes: Collagen II, Aggrecan and Sox9, which allows for their direct detection in cell cultures using immunocytochemical techniques [40][41][42][43] .Our analysis confirmed their presence.It was also shown that the levels of the identified markers varied depending on the passages (Fig. 2).This correlation of the decrease in the number of identified protein products with successive passages in the chondrocyte culture was observed by RT-PCR gene expression analysis and the presence of gene protein products.There was a high level of gene expression after the first passage (P1), which decreased in the two subsequent passages assessed-P2 and P3 (Figs. 3, 5), as confirmed in the literature 44,45 .This demonstrates susceptibility to dedifferentiation in later passages and reduced proliferative capacity of chondrocyte cultures.
Of key importance in our study was the fact that the obtained cell cultures showed the ability to proliferate after transplantation into the sternotomy wound.It has been previously shown that chondrocyte cells can be transplanted into knee joints and heal the injured area 46 .The cells actively proliferated and formed an extracellular matrix 43 .This effect was also observed in the sternal tissue we studied.Immunohistochemical and histopathological analysis showed the presence of chondrocytes in the formed wound (Figs. 6, 7, 8).They show typical morphological features of chondrocytes and the presence of characteristic protein products, i.e.Collagen II and Aggrecan.These products were observed both 3 weeks and 6 weeks after implantation.
To the best of our knowledge, the application of allogeneic chondrocytes to a sternal wound has been used for the first time in the treatment of a median sternotomy wound.This method appears to have great potential, as it rapidly fills the surgical wound, thus closing the space for the penetration of infection-causing microorganisms.Infections of deep sternal wounds and their long healing time remain a serious problem, contributing to prolonged hospitalization of patients, increasing treatment costs, and worse, being one of the life-threatening factors.
It is possible to obtain a viable culture of porcine chondrocytes from the myeloid process, which is influenced by the type of serum supplemented.The isolated cells show phenotypic stability up to 3 passages after isolation.Application of the cells in tissue glue directly to the sternotomy wound does not cause inflammation or necrosis, which allows us to conclude that this method is safe.We believe that the use of chondrocytes in tissue glue as an innovative method of treating sternotomy wounds will accelerate research and bring us closer to reducing infection of sternotomy wounds.
Nevertheless, our study has several limitations.Firstly, the animals used were completely healthy with no risk factors such as obesity, diabetes or age, that could affect the rate of wound healing.In addition, the animal model used moves on four limbs (front and back limbs), which causes a greater load on the sternum after surgery, which has no correlation with the post-operative situation in humans.Moreover, the metabolism and biological processes in animals are much faster than in humans, so it is impossible to accurately calculate whether the observation period we chose was valid.This requires further evaluation using older animals and additional time points.
Further studies evaluating the presence of chondrocytes implanted into the sternotomy wound at longer time points are needed to assess cell maturity and quantify the continued effectiveness of the method.

Animal studies
This part of the project was carried out at the Center for Cardiovascular Research and Development of American Heart of Poland.All methods were performed in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals".All procedures were approved by the Local Ethics Committee at the Medical University of Silesia in Katowice (Decision No. 61/2018).All methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).A total of 19 domestic swine (Sus scrofa domesticus), with an average body weight of 40 kg, were incorporated in this study.Animals were divided into the control and study groups for 3 and 6 weeks of observation (Fig. 9).

Anesthesia, monitoring and emergency procedures
In order to maintain general anesthesia, Isoflurane (Aerrane, Baxter, Poland) was used at a concentration of 1-3% in a mixture with oxygen (50-100%) or alternatively, a continuous drip infusion of Propofol (Braun, Poland) was administered in an amount of approx.12-20 mg/kg/h.During the procedure, basic physiological parameters such as ECG, arterial pressure, respiratory frequency and saturation were constantly monitored.Additionally, at the discretion of the anesthetist, the following were also administered: Fentanyl (Fentanyl WZF-50 µg/mL, WZF, Poland) at a dose of 2-4 µg/kg b.w IV by bolus or infusion at the rate of 0.03-0.06µg/kg/min.

Median sternotomy
After achieving a proper level of anesthesia, the surgical area was roughly cleaned and disinfected.Midline incision of the skin and subcutaneous tissues was performed and the sternum was exposed.With the help of an electric saw, the sternum was incised over the midline.The wound remained open for 60 min.In the control group, the sternum was closed with surgical wires, and the skin and subcutaneous tissues were closed with sutures.The study group received the medical glue with allogeneic chondrocytes on the borders of the sternum's wounds, the sternum was closed in the same manner as the control group, and another layer of medical glue with allogeneic chondrocytes was applied over the junction in the wound.The skin and subcutaneous tissues were closed in the

Cell culture
To optimize chondrocytes cell culture conditions, cells were seeded in four cell culture media.Each media contained the same among of antibiotics (Penicillin 10,000 U/ml, Streptomycin 10 mg/ml, Amphotericin B 25 μg/ ml), Vitamin C, DMEM/F12.Each culture media were prepared using different types of serum:10% FBS, 10% commercial pig serum (pig com), 10% self-prepared pig serum (self-pig) and FCS.Chondrocytes were seeded at a density 1 × 10 6 cells/T-75 flask in four different culture media.Cells were cultured until third passage, samples were taken for analysis after each passage.

Cells proliferation assay
Cells were seeded at a density 1.5 × 10 4 cells per cm 3 and incubated 24 h in 37 ºC, 5%CO 2 .The cell proliferation assay was performed according to the manufacturer's instructions (Promega, Wisconsin, United States).The measurement was performed on a day 0 (control), 1, 2 and 3 in 96-well TPP™ plate (PerkinElmer, Waltham, MA, USA).The intensity of the fluorescence emission was detected at 590 nm using a VICTOR™ Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA) with a 560 nm excitation source.

RNA Isolation and Quantitative RT-PCR
Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Germany) according to the manufacturer's protocol.cDNA was synthesized from 2 µg RNA with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions.Relative expression levels were measured in triplicates in a Roche Light Cycler 480 using Power SYBR Green PCR Master Mix (Applied Biosystems, Germany), 300 mM primers (Supplementary Materials; Table S3) and 1/15 cDNA stock.Values were calculated using the Pfaffl method and normalized to those of GAPDH.

Statistical analysis
Statistical analyses of the qRT-PCR data were performed with Microsoft Excel software.Normalized relative expression levels were used to calculate the mean and the SEM of all experiments, represented by columns and error bars on the figures, n = 3.

Figure 1 .
Figure 1.Swine chondrocytes morphology.Cells were cultured in four different media serum: Fetal bovine serum (FBS), Fetal calf serum (FCS), self-prepared pig serum and commercial pig serum.Cell morphology was analyzed at the same time points.Images were taken under an inverted light microscope (Delta Optical, Belgium).Scale bars = 100 µm.

Figure 2 .
Figure 2. Immunocytochemical analysis of Sox9, Collagen II and Aggrecan expression in swine chondrocytes.Representative pictures show the expression of surface markers in all tested serum (FBS, FCS, commercial Pig serum and self-prepared pig serum) in four passages (0, 1, 2, 3).Nuclei were stained with DAPI.Pictures were taken under a fluorescent microscope (Nikon ECLIPSE Ti, USA).

Figure 3 .
Figure 3. Identification of Sox9, Collagen II and Aggrecan in swine chondrocytes cultured in different serum at passage 0, 1, 2 and 3.In all tested serum media we identified proteins of interest.The blots were cut before hybridization with antibodies.GAPDH was used as a loading control.Original blots are shown in the Supplementary Materials; Fig. S1.

Figure 4 .
Figure 4. Cell proliferation assay.The X-axis indicates the variant of the serum used on the following days (1, 2 and 3).The Y-axis indicates relative cell survival.Values are mean ± SD (n = 3).

Figure 5 .
Figure 5. RT-qPCR expression analysis of Sox9, Collagen II and Aggrecan in swine chondrocytes.Gene expression was evaluated at different time points: at passage 0, 1, 2 and 3. GAPDH was used as loading control.Values are mean ± SEM (n = 3).

Figure 6 .
Figure 6.Immunodetection of Aggrecan and Collagen II expression in the extracellular matrix and cytoplasm of chondrocytes in the caudal part of the sternum.Representative samples stained with anti-Aggrecan antibody (1-4) and anti-Collagen II antibody (5-8) are shown.Similar results were obtained in cranial and medial parts of the sternum.Sections obtained from porcine xyphoid cartilage were the positive control.Scale bars represent 50 μm.

Table 1 .
Blood analysis in the study group after 3 and 6 weeks of observation.

Table 2 .
List of assessed histopathological parameters.

Table 3 .
Antibodies used for immunofluorescence staining.