For that reason, we established, in the cervical carcinoma cell line SiHa, a CDV resistant cell subline by stepwise dose escalation of CDV. We investigated the in vitro and in vivo phenotyping and development fee of SiHaCDV in contrast to parental cells. Also, we evaluated the vary ential gene expression profiles concerning SiHaparental and SiHaCDV by microarray evaluation so that you can recognize genes altering expression on collection of cells for CDV resistance. In the existing review, we centered around the examination of functions and pathways involved within the in flammatory response that transformed in SiHa cells comply with ing acquisition of CDV resistance. Importantly, we also examined whether SiHa cells that acquired resistance to CDV have been impaired in pathogenicity from the xenograft model. Results In vitro phenotyping of SiHaCDV SiHa cells were picked for CDV resistance following constant in vitro publicity on the drug for approximately 45 passages.
The resulting SiHaCDV presented a decreased development rate compared to SiHaparental. A stable CDV resistant phenotype while in the absence of selective drug strain was found for that SiHaCDV. When evaluated regarding cell development inhibition, a fold resistance selleck chemicals Oligomycin A of 100 against CDV was determined after 7 days of incubation using the drug. SiHaCDV displayed 10 fold cross resistance on the cytosine analogue Ara C and also to two unrelated ANPs. It should really be noted that the antiproliferative results of CDV for SiHaparental were time dependent, pointing to a unique mechanism of antiproliferative effects for these medication, in agreement with our prior report. An inhibition of 93% and 11% within the variety of cells was afforded by CDV treatment method at 158. seven uM for 7 days. To compare CDV effects on in duction of apoptosis in these cell cultures, annexin V and PI staining was performed.
Annexin V stains phos phatidylserine, a negatively charged phospholipid that’s translocated through the inner leaflet of your plasma mem brane to your outer leaflet for the duration of early apoptosis. Since PI isn’t going to enter into cells with intact membranes, it was used to identify necrotic cells. SiHaparental treated with CDV for seven days showed increased percentage of apoptotic cells and diminished quantities of viable cells. In contrast, SiHaCDV had been Vandetanib VEGFR inhibitor completely refractory to CDV induced apoptosis while they had been nonetheless able to react to PMEG, albeit to a reduced extent compared to SiHaparental. No signs of cell death by necrosis have been witnessed in any from the two cell lines following remedy with both CDV or PMEG. Differentially expressed genes upon acquisition of CDV resistance Gene expression profiling by microarray was carried out to identify probable mechanisms linked with CDV resistance.

Consequently, a number of RAS effector pathways that regulate fundamental biological processes such as proliferation, apoptosis, and cell motility, turn out to be activated andor deregulated. Even more especially, mutant KRAS disrupts actin cytoskeleton and maintains motility in colon cancer cells. Likewise, BRAF, a significant down stream effector of KRAS, can be regarded an oncogene whose activating mutations seem in 70% of human malignant melanomas and in about twelve 18% of human colon cancers. The most frequent BRAF mutation is at codon 600 that effects in elevated kinase action. Mutant BRAF may also interfere with organization of cytoskeleton and influence cell migration and invasion capability. Important techniques in invasion and metastasis are tightly regu lated or influenced from the Rho household GTPases, which could incorporate alterations in cell adhesion, cell matrix, cell cell interactions and actin organization, in the long run leading to the acquisition of an invasive phenotype.
Quite a few research have investigated the purpose of Rho GTPases in tumour progression exhibiting their contribution in cancer initiation and progression, through the acquisi tion of uncontrolled proliferation, survival and escape from apoptosis at the same time as tissue invasion and also the estab lishment of metastasis. Unlike KRAS and BRAF, mutations in RHO genes are tremendously rare Entinostat MS-275 in tumours, but their expression andor action is commonly altered in a variety of human cancers. RhoA is frequently above expressed in cancer, even though depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and inva sion in carcinoma cells. Another Rho family gene, Cdc42 can be essential for cell motility and in a position to induce a mesenchymal amoeboid transition in mela noma cells.
Regulation of Rho GTPases is exten sively studied and it really is nicely regarded that extracellular signal regulated kinase signaling is significant for cell motility through Rho GTPases. PI3K pathway can be involved in Rho loved ones signal transduction and influences properties like cell migration. While a significant Amonafide variety of research have analysed the purpose of Rho pathways in RAS induced transformation, rather little is regarded concerning the differential regulation of Rho GTPases by RAS and BRAF oncogene, at the same time as their subsequent contribution in oncogene distinct cell migra tion properties. To be able to invade into other tissues, epithelial cancer cells will have to disrupt the integrity of epithelium and base ment membrane to enter the underlying stroma. This in most cases needs acquisition of the migratory phenotype, a practice frequently referred as epithelial to mesenchy mal transition. Invasive epithelial cancer cells often demonstrate reduced expression of E cadherin, a cell cell adhesion protein, and an improved expression of mesenchymal markers, this kind of as vimentin and N cadherin.

Considering that HGEC permanently grown in 25 mM glucose show almost totally suppressed Pc amounts, compared to HGEC exposed to 5 mM glucose, we examined regardless of whether this modify may be attributed to dysregulation in the podocytic phenotype, earmarked by enhanced vimentin expression. Western blot examination demonstrated that vimentin expression was upregulated in HGEC,25 mM. Elevated vimentin expression levels were established following six weeks of culture in 25 mM glucose. Vimentin expression reached maximal ranges following 18 weeks of culture in 25 mM glucose, suggesting that modulation in the podocytic qualities occurred progressively with time. As a way to establish the time factors at which al terations in expression ranges of vimentin, also as other significant proteins expressed in podocytes oc curred, HGEC were exposed to 25 mM glucose for 1, two, four, six, 18 weeks.
From this time program an early time level plus a late time level were picked as a way to investigate no matter whether glucose effects have been re versible. The early time stage was picked since it sig nifies upregulation of your mesenchymal marker vimentin as well as late time level was chosen since alterations had been maximal. selelck kinase inhibitor Accordingly, we then examined irrespective of whether the observed transform in vimentin expression could be restored to typical ranges, with the early time level along with the late time level. HGEC exposed to 25 mM glucose for 6 weeks were reverted to normal glucose ranges for one more four weeks. Moreover HGEC,five mM to 25 mM 18w had been cultured in 5 mM glucose for four even more weeks. In each time intervals, early and late, vimentin reverted to reduce, usual ranges of expression. In vitro culturing of HGEC while in the presence of higher glucose amounts resulted in long term downregulation of CD10 CALLA protein expression We examined the expression of CD10 CALLA in a simi lar manner to vimentin.
FACS examination showed that HGEC,5 mM expressed CD10 CALLA. For the contrary, HGEC,25 mM demonstrated signifi cantly lowered cell surface related amounts. Appreciably lowered CD10 CALLA surface ranges had been established following 2 weeks of culture in 25 mM glucose and remained downregulated following six and 18 weeks of culture in 25 mM glucose. CD10 CALLA surface PF-2341066 structure levels remained considerably diminished following reverting glucose concentration to five mM for 18 weeks. We subsequent examined no matter if the observed downregulation of CD10 CALLA can be reversed at early and late time intervals, in HGEC sequentially grown in five mM glucose. In each situations CD10 CALLA remained diminished suggesting everlasting glucose induced downregulation of its expression. Reversible phenotypic modifications of expression in cultured podocytes are accompanied by ordinary levels of cell surface associated nephrin HGEC,25 mM displayed severely reduced nephrin expres sion in comparison to HGEC,five mM.

To tackle this question we implemented a siRNA method to in hibit Flt 1 expression in LoVo PlGF cells. We now have experimented with the Western blot for Flt 1, yet, we could only detect the above expressed a single but not the en dogenous one particular. This may perhaps be because of the lower expression degree of endogenous Flt 1. Thus, we could only show the data by quantitative PCR. The migration potential decreased when Flt 1 ranges were decreased by siRNA. This information signifies Flt one is required for PlGF induced CRC cell migration. To even further verify this result, we checked the impact of siFlt 1 on p38 phospho rylation in LoVo PlGF cells. Indeed, downregulation of Flt 1 considerably attenuated the phosphorylation of p38 in LoVo PlGF cells. We also located that the two migration and invasion ability decreased in LoVo PlGF cells when the PlGF was knocked down by utilizing the siRNA strategy.
Tumor progression was enhanced in LoVo PlGF cells ex vivo To confirm the role of PlGF in CRC ex vivo, tumor xenograft assays were carried out. During the observa tion period, three of the 4 LoVo PlGF cells im planted mice had palpable nodules as early since the 3rd week, and all of them had measurable tumors through the finish within the 14 week experimental time period. In contrast, only two on the 4 mice within the LoVo pcDNA group had palpable nodules, selleck chemical detectable only while in the 10th week. The LoVo PlGF group also acquired fat slower compared to the management group. Your body excess weight big difference became even more sizeable all through follow up. Mice have been sacrificed following week 14. The two the tumor radius and tumor volumes were bigger from the LoVo PlGF group. PlGF expression was without a doubt drastically greater during the tumor tissue induced by LoVo PlGF cell implantation. LoVo PlGF induced tumors had greater vascularity, greater microvessel density, and significantly less caspase three staining compared to the handle group.
Large expression of PlGF and Flt 1 in CRC tissues predicts worse prognosis We even more analyzed a publicly on the market gene expression dataset through the GEO database. In this cohort, higher PlGF and Flt one mRNA expressions have been observed in stage III IV ailments compared to stage I II illness. Org-27569 Individuals that had substantial Flt 1 and higher PlGF expression had shorter survival. Flt 1 expres sion was correlated with PlGF. This outcome supports the in vitro review effects, validating the results from NTUH cohorts, and strongly implies that higher PlGF amounts combined with higher Flt one expression boost cancer in vasion and lead to shorter survival. Discussion In this study we demonstrated that CRC cells express PlGF and Flt 1 have greater invasion migration skill. PlGF greater the invasion migration ability of colorec tal cancer cells by raising the phosphorylation of p38 MAPK and upregulating MMP9 expression. Overexpression of PlGF decreased the apoptosis mildly, but didn’t have an impact on the cell proliferation status.

Genetic and biochemical information have demonstrated that E6 and E7 facilitated beta catenin nuclear accumulation. These getting indicated that there is an activated Wnt B catenin signaling cascade in HPV associated premalig nant lesions that plays an effective position in accelerating cervical carcinogenesis. Activation within the Wnt pathway acted as secondary occasions which have been important for malig nant transformation of HPV contaminated epithelial cells. It’s also relevant to mention that detrimental regulators of Wnt signaling are epigenetically repressed in addition to a recent report clarifies an association between DKK3 and SFRP2 promoter methylation in cervical cancer. Notch Signaling As mentioned during the introductory segment that E2 functions like a repressor on the viral upstream regulatory area promoter that drives transcription of your E6 and E7 oncogenes, therefore loss of E2 can be a prerequisite for in creased E6 E7 expression.
To recognize if the inhib ition of E6 E7 expression by activated Notch1 takes place in the degree of URR promoter activity, HeLa cells had been transi ently transfected that has a plasmid to the URR promoter and an expression vector for activated Notch1. It was noted selleckchem that URR promoter activity was considerably re duced by cotransfection from the activated Notch1 expres sion. In addition it was observed that Notch one repressed URR by inhibiting selleck chemicals AP 1. Notch1 inhibited c Fos protein and concurrently enhanced one other Fos relatives member, Fra 1. Fra 1 lacked a transcription activating domain and acted as being a suppressor rather than an inducer of AP one dependent transcription. The information acquired by means of electrophoretic mobility shift assays indicated that Notch overexpression was correlated to altered AP 1 DNA binding activity and complicated composition.
After inducing a moderate degree of Notch ex pression, an enhanced DNA binding was demonstrated byAP 1. Nonetheless cells transfected with high expression amounts of Notch displayed a reduce in cFos signal and a rise in Fra1 signal. It is actually convincing to note that explants of HaCaT cells co expressing Jagged1and E6 E7 generated tumors better than 90 mm3. Nevertheless, co expression of Delta1 and E6 E7 created lesions of less than ten mm3. It was noted that Jagged 1 and E6 E7 co expressing cancer cells utilised PI3K Akt signaling axis to in duce EMT. Far more thorough insights suggest that Jagged one induced HES one that repressed Manic Fringe. These HES1 binding internet sites have been found at nucleotide position250 upstream in the transcriptional start out web-site of Manic Fringe. Notch one is also indicated to behave differ ently as HPV contaminated cells use Notch one throughout the professional gression from cervical intraepithelial lesions to invasive cervical carcinoma. Inducing apoptosis in HPV beneficial cancer cells Cellular and molecular scientific studies have outstandingly clari fied present concepts of part of HPV in cervical cancer.

Latest studies have reported that expression of spinal COX two mediates mechanical inflammatory discomfort hypersen sitivity, that’s decreased via the intrathecal injection TENS is usually a non pharmacologic and noninvasive treatment method for discomfort, usually utilized in patients with acute and chronic soreness. TENS has been proven for being effective for osteoarth ritis, rheumatoid arthritis, and postoperative pain and may relieve mechanical allodynia in animal designs of joint, muscle, and cutaneous irritation. TENS was applied with various frequencies, from 2 Hz to a hundred Hz and different frequencies led to numerous an algesic effects. During the research, the effect of TENS with alternating frequencies on inflammatory discomfort induced by CFA injection was evaluated. Our findings that TENS creates analgesic effect at six h just after CFA injection are consistent with prior findings that TENS partially reversed the hyperalgesia at 4 h right after carrageenan induced paw irritation.
In spite of TENS meditated analgesia, we did not detect an anti inflammatory impact, suggesting that TENS may inhibit the inflammatory pain hypersensi tivity independent of its anti inflammatory action. Our preceding examine has informed that EA, at the ST36 acu point, inhibited the expression of p ERK1 2 and p p38 MAPK in ipsilateral SCDH, and also induced a hyperalgisic response. These results full report advised the modu lation of MAPK activation in SCDH as an underlying mec hanisms of EA mediated inhibition of pain. Primarily based on latest literature, the essential mechanisms of TENS and EA mediated analgesia are comparable, however, the effects of TENS on ERK1 two activation stay unknown, notably on the spinal degree. In the existing research, our findings verified that, in addition to the modulation of PWTs, TENS treatment method considerably decreased the expression ranges of p ERK1 2 and COX 2 in SCDH at six h just after CFA injection.
Earlier research with the spinal level have proven that TENS mediated reduction of discomfort hyperalgesia is regulated through the release of gama aminobutyric acid and decreaed glutamate levels, along with endogenous opioid signaling. Furtermore, TENS mediated reduction of hyperalgsia by reducing the sensitization of dorsal horn neurons th rough regulating GABA and glutamate receptors. Glu tamate transmission Saracatinib via NMDA receptors was proven to become important for ERK1 two activation in SCDH neurons and its contribution to central sensitization. Furthermore, neu ronal expression of COX two from the spinal cord facilitated the advancement of the central component of inflammatory pain hypersensitivity through raising neuronal excitation and cutting down inhibition. General, regulation within the ERK1 2 COX two pathway in SCDH may be the signaling transuda tion pathway underlying the TENS mediated analgesia.

Inoue et al. demonstrated that the two CAPS and acidification generated elevations within the intracellular calcium concen tration in cultured human epidermal keratinocytes that might be inhibited through the TRPV1 antagonist, CapZ. Similarly, treatment method of human skin fibroblasts with CAPS induced vital modifications within the membrane latest as well as intracellular calcium degree that have been an tagonized by CapZ. It is consequently clear that TRPV1 is expressed and practical far from sensory nerves. Physiological or pathophysiological results of non neuronal TRPV1 are implicated in irritation, infection and immunity, the cardiovascular procedure and in circumstances such as weight problems. A physiological pathophysiological role for non neuronal TRPV1 is maybe nowhere a lot more obvious than from the situation of irritation, infection and immunity.
Although the effects of these TRPV1 channels are non neuronal, its evi dent they may well well effect indirectly on soreness and or neurogenic irritation. Keratinocytes functionally express TRPV1. These cells perform a significant function in sustaining the integrity of your immune response in skin likewise selleckchem as stimulating cutaneous inflammation by means of pros tanoid and cytokine release. TRPV1 activation by CAPS causes a rise in COX2 expression in human keratinocytes having a concomitant enhance in PGE2 levels in vitro. A rise in IL eight is additionally observed Stimulation of inflammatory mediator release by TRPV1 agonists from keratinocytes could well possess a significant result upon sensory nerves that have a high density in skin. Nonetheless, there exists accumulating proof that TRPV1 has functional roles away from sensory nerve activity. At the same time as keratinocytes, peripheral blood mono nuclear cells can also be directly affected by TRPV1 activation.
Such as, PBMCs undergo apoptosis when stimulated with CAPS or RTX, an result that’s reversed from the TRPV1 antagonist, AM630. You will find diverse other cells concerned in immunity that reply to TRPV1 and TRPA1 activation together with bone marrow derived dendritic cells, wherever CAPS prospects to dendritic cell matur ation and a rise more hints in antigen presentation. TRPV1 is expressed, practical and active inside of cells pertinent to irritation, infection and immunity. It is actually clear through the scientific studies that TRPV1 at least plays a para doxical position in inflammation in vivo, for instance, ex acerbating irritation in arthritis and still in experimentally induced sepsis, TRPV1 null mice demon strate elevated levels of pathological markers in compari son with wild style mice. It can’t be ruled out that this is because of differing results of neuronal and non neuronal TRPV1 channels. TRPV1 are actually proven to regulate vascular responses either by the effectively established neurogenic response that is mediated by sensory nerves or by way of a direct effect on vascular tissue.

The lack of established traditional of care treatment options for canines also presents an opportunity to evaluate novel therapies and protocols in topics with less sophisticated, non refractory disorder, prospects that happen to be difficult to unattainable in human patients. Osteosarcoma is definitely an great disorder candidate for inter species investigation of personalized medication ap proaches. It has been proven that canine and human OSA are analogous at a number of levels, histologically, behav iorally, genetically and with regards to response to therapy. The incidence of OSA in dogs is twenty fold better than in humans, with around 10,000 canines diagnosed annually when compared with approximately 2,650 key bone tumors in people, as a result rising the number of topics that happen to be out there for recruitment into clinical trials. OSA takes place generally at all around seven 9 many years of age, with big and giant breeds acquiring a 60 fold better possibility of establishing OSA.
Following amputation alone, 90% of canines die inside of a year, with reason behind death becoming associated to your advancement of metastasis, typically to your lung. Adjuvant hop over to this site chemotherapy can even more improve survival from 103 175 days following surgery alone, to 262 450 days. Even taking into account these dramatic improvements in survival time, the long lasting prognosis for OSA is morose and two year survival continues to be measured at amongst 10 26%. It truly is the poor long lasting survival of canines with OSA, alongside the translational value for that corre sponding human sickness, which helps make this tumor an ideal candidate for the identification of novel therapeutic agents employing PMed approaches. In this report we outline the results of a 20 topic feasibility review in canine osteosarcoma, together with the crucial aim of establishing the infrastructure and logistics to get a subsequent prospective substantial scale PMed trial.
The style of this examine was not intended to validate the clinical utility of a PMed report in canines with cancer. We describe the utility of worldwide gene expression profiling of osteosarcomas from canine individuals, which in Bafilomycin parallel with advances in laboratory procedures, bioinformatics tools and a physician reporting interface permits the application of real time genomic medicine during the con text of veterinary medication. Gene expression profiling is known as a instrument that has been made use of by other groups to examine canine osteosarcoma to identify differentially expressed genes that can stratify patients as short or long-term survival and identify biomarkers and pathways as sociated with patient prognosis. The PMed process utilized here is an assembly of 5 predictive methodologies that rank the overall drugs pre dictions weighted by the quantity of approaches which predict the drug, frequency of inclusion and strength of prediction.

The present study utilized the nomenclature of chemokines accredited by the IUIS WHO Subcommittee on Chemo kine Nomenclature. The mRNA levels of a panel of 43 chemokines and 19 chemokine receptors had been evaluated for each of the 4 cell lines. According to a Web Based PCR Array Information Analysis protocol offered by SABiosciences, the absent, lower and higher ex pression levels of chemokines had been defined as 35, 30 35 and 30 typical threshold cycles, respectively. OVCAR three cells extremely expressed. Except for CCL28, all were responsive to EGF or TNF. Although control CCL2 and CXCL16 cytokines had been expressed at lower amounts, they had been the two hugely ele vated following the addition of EGF or TNF. The results on CCL2, CCL20 and CXCL8 levels appeared to become syn ergistic when both EGF and TNF were added. Though CXCL6, CXCL10 and CX3CL1 had been induced by EGF or TNF, total they’d a lower ex pression.
OVCAR three cells displayed absent or lower chemokine receptor amounts. Even though CXCR4 was re sponsive to EGF or TNF, the expression ranges submit addition of both component, had been still lower. On top of that, EGF activated Akt and Erk whereas TNF solely activated I?B in OVCAR three cells. SKOV three cells expressed high levels of, of those, CXCL1 and CXCL8 had been responsive to both EGF or selleckchem TNF. Despite the fact that CCL20, CXCL2 and CXCL3 had been expressed at minimal levels in the absence of EGF or TNF, they were tremendously expressed by EGF and TNF. Depending on the fact that the level of CCL20 and CXCL8 in SKOV 3 cells exposed to each EGF and TNF had been better than the sum of the amounts reached with both EGF or TNF alone, we judged that in these circumstances, there was a synergistic result of EGF and TGF in these cells. In SKOV three cells chemokine re ceptor ranges have been both absent or minimal and had no re sponsiveness to both EGF or TNF.
SKOV 3 cells had a constitutively high degree of phosphorylated Akt that did not appear for being influenced by the addition of EGF or TNF. Even so EGF activated Erk with out I?B phosphorylation whereas TNF very acti vated I?B and had a compact impact about the activation of Erk. CaOV 3 cells really expressed have been much more responsive buy Roscovitine to EGF than TNF. CXCL8 appeared for being synergistically elevated in the presence of both EGF and TNF. CaOV 3 cells displayed higher basal ranges of CXCR4 and CXCR7 that had been not responsive to either EGF or TNF. Although CXCR5 level was minimal, it was tremendously expressed in response to EGF or TNF. EGF activated I?B, Akt and Erk whereas TNF activated only I?B and reduced EGF activated Akt and Erk in these cells. Lastly, TOV 21G cells remarkably expressed and CXCL3 have been a lot more responsive to EGF than TNF. Even though baseline TOV 21G cellular CXCL8 values were expressed at lower amounts, it had been really expressed by EGF.

The measurement was carried out in duplicate. The mRNA expression of every enzyme during the C9 10 loaded disc was converted to a relative number representing the quantity of mRNA compared together with the C12 13 unloaded disc utilizing the two Ct procedure. Paraffin embedded disc tissue preparation Loaded and unloaded discs had been excised, fixed in 4% paraformaldehyde, decalcified in 10% ethylenediaminete traacetic acid, embedded in paraffin, sectioned from the mid sagittal plane at five um thickness, and ready for immunohistochemical examination. Immunohistochemistry Catabolic and anti catabolic genes The quantity of immunopositive cells of every NP and AF dissected about the center was counted in five random large power fields working with BZ 9000 microscope and evaluation application. beneficial staining was expressed being a percentage of immunopositive cells to complete cell population mea sured by counting the nuclei.
Aggrecanolytic activity Immunohistochemistry working with cleavage website exact anti bodies for aggrecan was carried out to detect aggrecan core protein fragments produced by MMP and aggreca nase activity. Just after deglycosylation with the aggrecan core proteins implementing chondroitinase ABC and keratanases, sections were taken care of at four C above night with one 20 hop over to this website diluted mouse monoclonal antibody BC 14, which recognized the N terminal neoepitope sequence of 342FFGVG created by MMP cleavage inside the intergrobular domain of aggrecan, or one one hundred diluted mouse monoclonal antibody BC three, which recognized the N terminal neoepitope sequence of 374ARGSV created by aggrecanase BMS599626 cleavage. The secondary antibody was a peroxidase labeled anti mouse antibody made use of at space temperature for 30 minutes. Brown shade improvement, counterstaining, cell counting, and detrimental manage preparation were carried out as described over.
Statistical examination Two way mixed style analysis of variance with all the Turkey Kramer submit hoc check was utilized to assess adjustments of mRNA degree for the results of disc level and time. Two way ANOVA using the Turkey Kramer submit hoc test was applied to evaluate changes of percentage of immunopo sitive cells similarly. Information analyses have been carried out making use of PASW Statistics 18. Statistical significance was accepted at P 0. 05. All values are expressed as suggest typical deviation. Success All animals tolerated surgery well and acquired physique bodyweight through the entire experiment 455 to 526 g at 7 days, 497 to 563 g at 28 days, and 543 to 614 g at 56 days. All springs maintained their compressive length and absolutely recovered without delay right after release, indicating sustained axial loading. No indications of infection, skin necrosis, neurological concerns, or instrument failure were observed.