Arrays for Growth Factor and Serum Cytokines Quantification

Background:  Wound healing deficiencies in diabetic individuals have been attributed to over 100 known physiological factors. Diabetes poses a major risk factor for periodontitis, with a threefold risk in patients diagnosed with diabetes compared to those without. Platelet rich fibrin (PRF) is a second-generation autologous platelet rich concentrate that is known to release growth factors and cytokines that  aid in wound healing. PRF membranes are utilized to accelerate the wound healing in dental and medical settings. Recent evidence demonstrate the benefits of utilizing PRF to treat chronic wounds or ulcers. The purpose of this study is to investigate the differences in the amount of growth factors and cytokines released from PRF between patients with non-regulated type 2 diabetes mellitus and systemically healthy participants, as well as to determine if platelet count influences the amounts of growth factors and cytokines released from PRF.

Hypothesis: There is a downregulation of the amounts of growth factors and cytokines (EGF, bFGF, IGF-1, VEGF-A, TGF 1, PDGF-BB, IL-1 , IL-6, TNF-, IL-8 and IL4) released from PRF, as well as the number of platelets in diabetic patients compared to non-diabetic individuals.

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M&M: A total of 43 ml of blood will be collected from non-regulated diabetic and non-diabetic patients. Forty ml will be centrifuged to be processed into PRF. The remaining 3 ml will be used to quantify complete blood counts. The collected PRF will be compressed, weighed and then immediately digested with trypsin for 24 hours. It will be used for the quantification of EGF, bFGF, IGF-1, VEGF-A, TGF 1, PDGF-BB, IL-1 , IL-6, TNF-, IL-8 and IL4 using protein antibody arrays.

Statistics:

10 subjects, per group will be included in this study. Summary statistics (mean, standard deviation, standard error, 95% confidence interval for the mean, minimum, maximum) will be calculated for the growth factors and cytokines by group. Two- sample t-tests will be used for between group comparisons for difference in growth factors and cytokines. Associations of the number of circulating platelets with growth factors and cytokines amounts released from PRF will be evaluated using scatterplots and correlation coefficients. A 5% significance level will be used for all tests.

Comparison of the released amounts of growth factors and cytokines from platelet-rich fibrin (PRF) from patients with and without type 2 diabetes mellitus

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1. Background and Rationale:

Diabetes mellitus has been reported to affect 170 million people worldwide, including 20.8 million in the United States and these numbers are projected to double by 2030 [1, 2]. Diabetes mellitus results in hyperglycemia due to defects in insulin secretion and/or insulin action which ultimately affects multiple organ systems. [3].

One of the complications of diabetes mellitus is impaired wound healing. Wound healing is a complex process involving the activation of various cells after injury including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets [5]. The coordination and maintenance of healing is facilitated by many growth factors and cytokines released by these cell types. Wound healing deficiencies in diabetic individuals have been attributed to over 100 known physiological factors, some of which include, a reduced or impaired growth factor and cytokine production, macrophage function, collagen accumulation, angiogenic response, quantity of granulation tissue, epidermal barrier function, migration and proliferation of keratinocyte and fibroblast migration, bone healing, number of epidermal nerves and remodeling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) [6-10].

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Diabetes mellitus is associated with various blood cell abnormalities, especially of white blood cells (WBC) and platelets. An increased WBC count was noted in the development of type 2 diabetes and as a predictor of the worsening of insulin action in a study conducted on Pima Indians [11, 12]. Increased clotting, impaired clot breakdown, endothelial dysfunction and platelet hyper-reactivity are some of the platelet abnormalities associated with diabetes. Platelet activation has also been shown to be reduced following improvement in metabolic control [11, 12].

Diabetes is also a major risk factor for periodontitis, with a threefold risk in patients diagnosed with diabetes compared non-diabetic patients. [13]. A bidirectional or “two-way” relationship between diabetes and periodontitis has received recent emphasis, in that not only is diabetes a risk factor for periodontitis but the severity of the periodontal condition could have a negative effect on the control of the glycemic index. Poor glycemic control was associated with severe periodontitis sustained after 2 years of follow up, thus implicating periodontitis as a risk factor for the inadequate management of diabetes [14].

Acute phase proteins such as C-reactive proteins (CRP) are induced by TNF-α and IL-6 and the two are also known to contribute to insulin resistance by impairing intracellular insulin signaling leading to the development of type 2 diabetes [15, 16].  Besides diabetes, serum levels of IL-6 and CRP are also elevated in patients with periodontitis with a positive correlation of IL-6 to the disease severity [17, 18].

Growth factors and cytokines may be used as an adjunct in various dental and medical treatment protocols to promote healing. One of the recently introduced treatment modalities for periodontal defects include the delivery of growth factors via an autologous blood concentrate in a liquid or gel form [21-23]. Platelet rich concentrates from autologous blood are commonly used in two different forms: platelet-rich plasma (PRP) and platelet-rich fibrin (PRF). Platelet-rich plasma is the first generation of the platelet-rich concentrates from whole venous blood and has been demonstrated to enhance clinical healing of the surgical sites as an autologous source of growth factors [21-23]. Choukroun et al. introduced the use of PRF which is considered as a second generation of platelet concentrates material. PRFs are compressed into a membrane form to be used in periodontal surgeries [21-23].

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Growth factors including PDGF, TGF-β1, VEGF, EGF and IGF have been demonstrated to be released by PRF [21-23]. PDGF stimulates mesenchymal cells and regulates their proliferation, migration and survival. PDGF also has a key role in enhancing scar formation along with TGF-. Osteoblasts or fibroblasts produce TGF- which plays a role in the synthesis of fibronectin and type I collagen [21-23]. IGF upregulates the proliferation and differentiation of most cell types and acts as a cell-protector. VEGF is one of the primary growth factors involved in the initiation of angiogenesis and directly influences the fate of endothelial cells. It is an omnipresent growth factor that promotes vascular growth and development [21-23].

As an autologous source of growth factors, the use of PRF eliminates the potential of disease transmission and immunogenic reactions. PRF has been increasingly used for ridge augmentations, sinus graft procedures, and regenerative therapies around the teeth [23-28]. The physical properties of PRF enables it to be used as a biologic scaffold for guided tissue regeneration in bony defects around teeth affected by periodontal destruction. PRF is preferred over PRP since the fibrin membrane of PRF has been demonstrated to more slowly release growth factors  and cytokines during wound healing [29, 30].

As previously stated, diabetes mellitus is associated with delayed wound healing and various therapeutic measures have been considered to combat this issue. The use of PRP and PRF have been recently implemented in the treatment protocol in the medical field especially for non-healing diabetic skin ulcers. Ding et al. [31] reported that PRF accelerated skin wound healing in diabetic mouse models and the result was attributed to increased angiogenesis. Another animal study on diabetic rabbits exhibited greater new bone formation in critical sized defects created in the calvaria treated with PRF compared to those without [32]. A prospective study utilized PRF as a healing aid for extraction sockets in diabetic patients [33]. The results showed that PRF utilization resulted in a faster healing rate and also reduced the need for antibiotics. Thus, the use of PRF following tooth extractions in diabetic patients may improve healing and reduce infection rates. A controlled study in fourteen diabetic patients with a mean HbAlc level of 9.5% using autologous platelet gel for the treatment of diabetic foot ulcers demonstrated accelerated wound healing [34].

The increased production of pro-inflammatory cytokines like IL-6, TNF- and IL-1 are commonly encountered in patients with chronic inflammatory conditions like diabetes and periodontitis. This production may lead to a downregulation of growth factors in the plasma [29].

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A recent study from our group evaluated the relationship between healthy patients and those with periodontitis to compare the amounts of growth factors released from PRF of such patients.

The influence of another chronic inflammatory disease, such as diabetes mellitus affecting the quantity and release of growth factors and cytokines from PRF has not been widely studied. Hence, the proposed study will be investigating the growth factors in PRF from patients with non-regulated type 2 diabetes mellitus, compared with systemically healthy participants, and to determine if the quantity of growth factors and cytokines released are affected by the number of circulating platelets.

 

2. Purpose:

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The purpose of this study is to investigate the differences in the concentration of growth factors and cytokines released from PRF between patients with non-regulated type 2 diabetes mellitus and non-diabetic systemically healthy participants and to determine if platelet count influences the amounts of growth factors and cytokines released from PRF in both groups.

3. Hypothesis:

Null hypothesis

1. There is no difference in growth factors and cytokines (EGF, FGF, IGF-1, VEGF-A, TGF-1, PDGF-BB, IL-1 , IL-6, TNF-, IL-8 and IL4) amounts released from PRF obtained from systemically healthy patients or patients with non-regulated type 2 diabetes mellitus.
2. There is no relationship between the number of circulating platelets to the growth factors and cytokines amounts released from PRF obtained from systemically healthy participants or patients with non-regulated type 2 diabetes mellitus.

Alternative hypothesis

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1. There is a difference in growth factors and cytokines content and amount released from PRF obtained from systemically healthy participants and patients with non-regulated type 2 diabetes mellitus.

• It is believed that patients with non-regulated type 2 diabetes mellitus will exhibit a down regulation of growth factors and cytokines amounts released when compared to PRF obtained from healthy participants, due to inflammatory responses.

2) There is a relationship between the number of circulating platelets to the growth factor and cytokines amounts released from PRF obtained from healthy participants or patients with non- regulated type 2 diabetes mellitus.

• It is believed that patients with higher number of platelets will exhibit more growth factor and cytokines amounts released from PRF from healthy participants and patients with non-regulated type 2 diabetes mellitus.

4. Materials and Methods:

Following review and approval of the protocol by The Institutional Review Board (IRB) of the Indiana University, this study will be initiated. Written informed consent and authorization will be obtained from all participants and they will be given a copy of the signed informed consent documents. The procedures performed in this study, including collecting blood samples from subjects, are for research purposes only and are not within the standard of care.

Recruitment of Subjects

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All participants will be recruited from patient clinics of Indiana University School of Dentistry. Subjects who agree to participate in the study will be screened to confirm that subjects meet inclusion/exclusion criteria. Written informed consent will be obtained by the participants prior to any data collection.

Study Group

Up to ten patients with diagnosis of non-regulated type 2 diabetes mellitus and a HbA1c > 6.5% will be recruited as subjects in the study group.

• Patients with a HbA1c > 6.5% determined by A1CNow^SELF CHECK*.

Control group

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The control group consists of up to 10 non-diabetic systemically healthy subjects.

• Patients with a HbA1c <5.6% determined by a A1CNow^SELF CHECK*.

(*A1CNow^ SELF CHECK: Pts Diagnostics, 7736 Zionsville Road, Indianapolis, IN 46268, USA)

Recruitment will be announced via email to all student dentists and residents at post graduate programs, and a flyer that is approved by the IRB will be posted inside IUSD for all patients of IUSD and for any students who know any subjects interested in participating in the study. This email will help the student dentists and residents to identify patients that satisfy the inclusion criteria and to recruit potential subjects. The patients who are interested in participating in the study will contact the PI or student PI, via telephone or email and will be screened at the Periodontics department for the final inclusion in the study. The nature of the study, risks, benefits and payment for participation will be discussed during the screening appointment.

Inclusion/Exclusion criteria

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The following inclusion criteria and exclusion criteria will be used for all participants.

Inclusion criteria

• Patients who agree to have their blood drawn for growth factor and cytokine analyses.
• Patients who agree to have their HbA1c level checked using the A1CNow^SELF CHECK*.
• Patients with a HbA1c > 6.5% as determined by A1CNow^SELF CHECK*.

• Age: >30 years-old
  Age range between study group and control group need to be matched. To achieve this recruitment may be limited to enrolling an initial group of 5 patients with diabetes who are matched with controls. Subsequently a second cohort of five patients with non-regulated diabetes will be enrolled followed by a matched set of control subjects.  This process will continue until the entire panel and subsequent controls have been enrolled.
• Male and Female: Gender between study group and control group need to be matched.
• Controlled systemic disease (Self-reported)
  (American Society of Anesthesiologists (ASA) Classification I and II patients: mild systemic disease without functional limitation)
• Non-smoker (Self-reported)

Exclusion criteria

• Patient with severe systemic diseases (ASA more than II), other than non-regulated type 2 diabetes mellitus)
• Class III obesity (BMI > 40 kg/m²) or underweight (BMI < 18.5 kg/m²)
• Pregnancy
• History of myocardial infarction, cerebrovascular accident, transient ischemic attack, coronary artery disease in the past 6 months.
• Immune compromised patient (HIV, AIDS)
• Smokers or alcoholic patients (Self report)
• Current chemotherapy or radiotherapy
• Current corticosteroid, anticoagulant, antiplatelet, NSAIDs or other anti-inflammatory drug use
• History of drug or substance abuse (Self-reported)

Clinical Procedures

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1. Screening and Consent

Following a regularly scheduled visit in which medical history is routinely reviewed, patients who are perceived to qualify for the study will be approached by the PI or student PI to determine interest in their participation in the study.  If interested, the patient will complete the consent process and be asked the inclusion/exclusion questions, including a self-reported HbA1c status, if known.  Data collected in Axium related to medical history and periodontal diagnosis may be recorded as study data.

2. Collection of blood sample and data collection

The participant will be seen at the graduate periodontics clinic at the same day of the regularly scheduled visit or at a later day as per the participant and PI’s convenience. To confirm that the participants meet the inclusion/exclusion criteria (as mentioned above), the BMI will be determined using the participant’s height and weight. The HbA1c level will be determined using A1CNow^SELFCHECK after obtaining the participant’s consent for the evaluation.

Intravenous access through right or left cubital region of arm or dorsal of right or left hand for blood collection for PRF will be processed by trained periodontists. The site selection for access to the vein will be determined by the examiners based on vessel size and accessibility. A total of 43 ml of blood will be collected. Three ml of the collected blood will be sent to a clinical lab to obtain a complete blood count (CBC) including platelet count. Compensation of $50 gift card (per participant) will be given to those subjects for allowing collection of blood samples as part of this research study.

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ASSESSMENT OF SAFETY

1. Study Safety Management

An adverse event will be considered any unfavorable and unintended sign, symptom or disease associated with study participation.  All adverse events will be reported to the IRB during annual review.

All serious adverse events will be reported immediately to the IRB. Serious adverse events include:  any event resulting in death, is life-threatening, requires hospitalization, results in disability/permanent damage, congenital anomaly/birth defect, or requires intervention to prevent permanent impairment or damage, and/or other medical events (31).

2. Medical/Dental Monitor

The principal investigator and student investigator will be responsible for overseeing the conduct of the study.

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3. Methods for Assessing Safety Parameters

Each subject will be given the student investigator’s phone number to call if they should experience any discomfort or unusual symptoms resulting from the blood draw.  Should an adverse event result from participation in the study, the PI and student PI will see that the subject is presented with options for follow up care.

Platelet-rich fibrin collection

PRF will be isolated with procedures described by Kobayashi [30]. Forty ml of intravenous blood collected will be distributed into 4 tubes of 10 ml each without the addition of any anticoagulant agents and centrifuged for 2700 rpm for 12 minutes (Clinifuge, Kendro Laboratory Products, Hanau, Germany). Each of the four tubes will provide one PRF clot.  The centrifugation will result in a fibrin clot being formed as a central layer in the tube between the acellular plasma above and the red corpuscles below. The collected fibrin clots will be compressed in a PRF box (OSUNG MND Co, Kyonggi-do, Korea) to form a flattened membrane. The contents of the PRF box include a lid that is used to compress the PRF clots against a mesh layer and a container to collect the serum extruded from the clots. All 4 of the fibrin clots will be compressed for 5 minutes to form 4 membranes, three of which will be weighed and frozen for future research to be conducted by the PI. For the purpose of standardization, the weight of the PRF membrane will be determined by weighing a 5 ml plastic vial containing 3 ml of saline, without the PRF membrane at first followed by weighing the same tube with the PRF membrane added to it at room temperature. The difference in weight between the two measurements will be recorded as the actual weight of the PRF membrane. Following the measurements of the weight of the PRF membrane, the saline will be replaced by 3ml of 1g/ml trypsin to initiate the digestion of the PRF membrane for 24 hours. The volume of the liquid exudate that is collected in the container of the PRF box will be measured and frozen for future investigations to be conducted by the PI.

Platelet number quantification

Platelet number will be determined using the 3ml sample of venous blood that will be sent to a clinical lab (IU Health Pathology Laboratory) for quantification via the complete blood count (CBC) test.

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Arrays for growth factor and serum cytokines quantification

The compressed PRF membrane will be digested at 37^oC in a 5 ml plastic vial containing 3 ml of 1 g/ml trypsin (Worthington Biochemical Corporation, NJ, USA) for 24 hours. 1 mg/ml soybean trypsin inhibitor (Worthington Biochemical Corporation, NJ, USA) will be used to stop the digestion process after 24 hours.  Following the inhibition of the trypsin, all the specimens will be frozen until subjected to the protein array tests. Cytokine protein arrays (RayBio Custom C- Series Human Antibody Array Kit, RayBiotech, Norcross,GA.) will be used to detect growth factors and cytokines including EGF, bFGF, IGF-1, VEGF-A, TGF 1, PDGF-BB, IL-1 , IL-6, TNF-, IL-8 and IL4, expressed from the PRF membranes. Since the cytokine protein array kit to be used will be customized, the arrangement of cytokines on the membrane will be determined by the company.

The manufacturer instructions will be followed for the detection of the growth factors and cytokines with the antibody array. The membranes will be blocked for 30 min, incubated for 3 h with the samples, washed, incubated for 2 h with biotin-conjugated antibodies, washed, and incubated with horseradish peroxidase-conjugated streptavidin for 2 h as per the manufacturer. Detection agents supplied in the array kits will be mixed and applied to each membrane for 2 min. The cytokines on the membranes will then be visualized by autoradiography on X-ray film. Signal intensities will be quantified with a Bio-Rad Gel Doc XR Imaging System and analyzed with Quantity One software (Bio-Rad, Life Science).

The samples will be run in triplicate independently for the PRF membranes in the test and the control groups.

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The ratio of each growth factor and cytokine from the diabetes group (test group) to the non-diabetic systemically healthy group (control group) will be determined in order to see increase, decrease or no change with respect to their expression levels.

5. Statistical Methods:

Summary statistics (mean, standard deviation, standard error, 95% confidence interval for the mean, minimum, maximum) will be calculated for the growth factors and cytokines by group. Comparisons between groups for differences in growth factors and cytokines will be made using two-sample t-tests. Associations of the number of circulating platelets with growth factors content and amount released from PRF will be evaluated using scatterplots and correlation coefficients. A 5% significance level will be used for all tests.

Based on the study by Kobayashi [30], the within-group standard deviations for the total growth factor release are 235 pg/mL for TGF, 63 pg/mL for VEGF, 95 pg/mL for EGF, 33 pg/mL for IGF, and 155 pg/mL for PDGF-BB. With a sample size of 10 subjects per group, the study will have 80% power to detect an effect size of 1.325, for differences between groups of 311 pg/mL for TGF, 84 pg/mL for VEGF, 126 pg/mL for EGF, 44 pg/mL for IGF, and 205 pg/mL for PDGF-BB, assuming two-sided tests each conducted at a 5% significance level.

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