Systemic Biochemical Signature and Functional Quality Index of Calcaneal Bone Marrow Aspirate in Adults Undergoing Foot or Ankle Surgery

This is a single-center, observational, prospective cohort study conducted at a university hospital in Brazil. The study will enroll 40 adults aged 40 to 80 years who are undergoing clinically indicated foot or ankle surgery. Participants may present with or without comorbidities, including type 2 diabetes mellitus and, when present, active Wagner grade 2-4 diabetic foot ulcers. These clinical characteristics will be recorded as observational variables and will not define intervention assignments or separate study arms. All participants will provide written informed consent before any study procedures.

Before surgery, participants will undergo a panel of routine laboratory tests covering four domains: inflammatory burden, including complete blood count, derived inflammatory indices, and albumin; glycometabolic burden, including fasting glucose, oral glucose tolerance test when clinically feasible, HbA1c, triglycerides, and cholesterol profile; mineral-renal burden, including creatinine, urea, ionized calcium, phosphorus, magnesium, alkaline phosphatase, parathyroid hormone, and 25-OH vitamin D; and tissue-energetic damage, including lactate dehydrogenase, AST, and ALT.

During the clinically indicated orthopedic surgery, calcaneal bone marrow aspirate will be collected from the same operative field. Mesenchymal stem cells will be isolated, cultured, and evaluated using standardized functional assays, including clonogenic capacity, osteogenic differentiation, adipogenic differentiation, cellular senescence, cytokine secretion, and cellular viability.

The Functional Quality Index of Bone Marrow Aspirate (IQF-BMA) will be constructed from standardized routine laboratory variables representing inflammatory, glycometabolic, mineral-renal, and tissue-energetic domains. The primary analysis will examine the association between the IQF-BMA and the clonogenic capacity of calcaneal bone marrow mesenchymal stem cells. Secondary analyses will examine associations between laboratory markers or the composite index and other cellular functional outcomes. Results will be reported primarily as effect estimates and 95% confidence intervals to support the planning of future, larger studies.

Primary outcomes

• Clonogenic capacity of calcaneal bone marrow mesenchymal stem cells (CFU-F) [Up to 14 days after bone marrow aspirate collection.]: Number of colony-forming unit fibroblasts (CFU-F) per milliliter of calcaneal bone marrow aspirate, quantified in primary culture under standardized conditions.

Secondary outcomes

• Osteogenic and adipogenic differentiation of mesenchymal stem cells [Up to 28 days after bone marrow aspirate collection.]: Osteogenic and adipogenic differentiation capacity of calcaneal bone marrow mesenchymal stem cells, assessed by alkaline phosphatase activity and semi-quantitative scoring of Alizarin Red and Oil Red O staining.

Sex: All, ages: 40 Years to 80 Years, accepts healthy volunteers

Inclusion Criteria

* Age 40 to 80 years.
* Scheduled for clinically indicated foot or ankle surgery at the participating university hospital.
* Ability to understand the study procedures and provide written informed consent.
* Willingness to provide preoperative blood samples and intraoperative calcaneal bone marrow aspirate according to the study protocol.
* Participants may have comorbidities, including type 2 diabetes mellitus and, when present, active Wagner grade 2-4 diabetic foot ulcers. These clinical characteristics will be recorded for observational analyses and do not define separate study groups.

Exclusion Criteria

* Critical limb ischemia without revascularization options or hemodynamic instability.
* Active systemic infection or sepsis at the time of enrollment.
* End-stage renal disease requiring dialysis.
* Current use of high-dose systemic corticosteroids or other immunosuppressive drugs.
* Current or recent malignancy within the last 5 years, except adequately treated non-melanoma skin cancer.
* Significant cognitive impairment or psychiatric condition that, in the opinion of the investigator, precludes informed consent or adherence to study procedures.
* Pregnancy or breastfeeding.

• Vanessa Bertolucci, PhD Candidate, Principal investigator, Universidade Sao Francisco