Table 3. Recommended assessments and measurements/assays for IND applications of cartilage cellular therapy products.

(A) General FDA guidance for medical devices, pharmaceuticals, and biologics. (B) Guidance specific for cartilage cellular therapy products. (C) Literature-based recommendations for cartilage cellular therapy products. ASTM, the American Society for Testing and Materials; H&E, hematoxylin and eosin; DMMB dimethylmethylene blue; GAG, glycosaminoglycan; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunoassay.

Assessment	Measurement / Assay	In vitro	In vivo	Refs
A. Selected preclinical assessments recommended by the FDA for medical devices, pharmaceuticals, and biologics
CMC safety and quality testing	Per requirements of 21 CFR 610, including potency, sterility, purity, identity, stability, mycoplasma, endotoxin, and other adventitious agents	×	-	(3, 126, 127)
Genotoxicity	Standard genetic toxicology. Measure chromosomal aberrations using in vitro and in vivo (for mutagenic assessment) tests	×	×	(3, 128, 129)
Carcinogenicity	Evaluation of carcinogenic and tumorigenic potential. Early stage in vitro assays (e.g., cell transformation) can be performed. In vivo studies are recommended (e.g., rodent study)	×	×	(3, 128, 130)
Toxicity	Chronic toxicity testing (in rodents and non-rodents)	-	×	(3, 128, 131)
Toxicokinetics, systemic exposure	Generation of pharmacokinetic data to describe the systemic exposure (in vivo) and its relationship to dose and the time course of the toxicity study	-	×	(128, 132)
Safety pharmacology	Identify, evaluate, and investigate the mechanism of any adverse pharmacodynamic effect of a therapy product (i.e. substance) that may have relevance to its human safety	×	×	(133)
Developmental and Reproductive toxicity (DART)	Assess the effect of a therapeutic product (i.e., pharmaceutical) on reproduction and development (if applicable). Complete life cycle (in vivo) studies are recommended. Alternative assays (e.g., in vitro, ex vivo) may defer or replace (in certain circumstances) conventional in vivo studies	×	×	(128, 134)
Immunotoxicity	Identify compounds which have the potential to be immunotoxic. Assays include T cell-dependent antibody response (TDAR), immunophenotyping, and cell/function activation assays	×	×	(128, 135)
Cytotoxicity	Agar diffusion cell culture screening	×	×	(41)
Degradation	In vivo degradation assessment recommended for absorbable therapy products		×	(41)
B. Cartilage-specific recommended assessments (from FDA and ASTM)
Mechanical data	Address the product’s ability to withstand loading, the fixation method, and the propensity to generate wear debris. Compressive testing is specifically recommended to quantify aggregate modulus (HA), shear modulus (μ), permeability (k), dynamic modulus (G*)	×	×	(2)
Biological response	Biological activity, including proof-of-concept (treatment feasibility) and safety data	-	×	(2)
Durability	Length of time needed to assess repair tissue formation and wear resistance. Large animal models recommended	-	×	(2)
Toxicology	Assessment of local and systemic toxicity	-	×	(2)
Dose response	Characterization of the therapy product’s components that may affect repair tissue formation. Large animal models recommended	-	×	(2)
Arthroscopy and/or MRI	Interim arthroscopic assessments and/or MRI evaluations in the animal studies (to reduce number of animals euthanized at each time)	-	×	(2)
Tissue structure	Histological evaluation (macroscopic and microscopic analysis and scoring)	×	×	(111)
General biochemical characterization	Biochemical quantification of proteins and proteoglycans in repair tissue compared to native cartilage is recommended but not specified	×	×	(111)
Part C. Recommended assessments for cartilage cellular therapy products (from scientific literature and guidance documents)
Compressive mechanics	Aggregate modulus, shear modulus, and permeability via creep indentation; or instantaneous modulus, relaxation modulus, and coefficient of viscosity via stress-relaxation	×	×	(136, 137)
Tensile mechanics	Young’s modulus, ultimate tensile strength, and toughness via uniaxial tensile loading	×	×	(138)
Tribology	Coefficient of friction (μ) via tribometer	×	×	(139, 140)
Mechanics of fixation	Fixation strength via pull-apart or push-through or lap shear tests	×	-	(141)
Mechanics of integration	Interface strength via pull-apart or push-through tests	-	×	(142)
Chromosome instability and genetic alterations	Karyotype analysis of cells (for extensively passaged cells and stem cell products)	×	-	(41, 143)
Tumor formation and cell malignancy	Soft agar colony formation, tumor sphere assay, colony forming/clonogenic assay, immunodeficient rodent tumorigenicity assay (for stem cell products)	×	×	(41)
Abnormal cell differentiation	FACS, qRT-PCR (for stem cell products)	×	-	(41)
Tissue structure	Histological staining for general structure and integration (H&E), collagen (picrosirius red), and glycosaminoglycan (safranin-O)	×	×	(144)
Biochemical composition	Collagen (e.g., hydroxyproline) Glycosaminoglycan (e.g., DMMB Blyscan GAG) DNA (e.g., Picogreen) Crosslinks (e.g., mass spectrometry or HPLC-based)	×	×	(145–148)
Activity	Pre- and post-operative activity measurement via activity monitors	-	×	(149)
Repair tissue collagens	Collagen types via mass spectrometry or immunohistochemistry or ELISA (at least types I and II)	-	×	(148)
Midpoint assessments	Arthroscopic evaluation, synovium analysis	-	×	(66)
Animal observation	Lameness, behavior, and eating and drinking habits	-	×	(82)
Additional terminal assessments	Necropsy, gross pathology, histological evaluation and scoring, synovium analysis, immunohistochemistry of immune cells	-	×	(82)