Anisotropic hydrogel scaffold by flow-induced stereolithography 3D printing technique.

Essential organs, such as the heart and liver, contain a unique porous network that allows oxygen and nutrients to be exchanged, with distinct random to ordered regions displaying varying degrees of strength. A novel technique, referred to here as flow-induced lithography, was developed. This technique generates tunable anisotropic three-dimensional (3D) structures. The ink for this bioprinting technique was made of titanium dioxide nanorods (Ti) and kaolinite nanoclay (KLT) dispersed in a GelMA/PEGDA polymeric suspension. By controlling the flow rate, aligned particle microstructures were achieved in the suspensions. The application of UV light to trigger the polymerization of the photoactive prepolymer freezes the oriented particles in the polymer network. Because the viability test was successful in shearing suspensions containing cells, the flow-induced lithography technique can be used with both acellular scaffolds and cell-laden structures. Fabricated hydrogels show outstanding mechanical properties resembling human tissues, as well as significant cell viability (> 95 %) over one week. As a result of this technique and the introduction of bio-ink, a novel approach has been pioneered for developing anisotropic tissue implants utilizing low-viscosity biomaterials.

Manipulating mechanical properties of PEG-based hydrogel nanocomposite: A potential versatile bio-adhesive for the suture-less repair of tissue.

Multifunctional bio-adhesives with tunable mechanical properties are obtained by controlling the orientation of anisotropic particles in a blend of fast-curing hydrogel with an imposed capillary flow. The suspensions' microstructural evolution was monitored by the small-angle light scattering (SALS) method during flow up to the critical Péclet number (Pe≈1) necessary for particle orientation and hydrogel crosslinking. The multifunctional bio-adhesives were obtained by combining flow and UV light exposure for rapid photo-curing of PEGDA medium and freezing titania rods' ordered microstructures. Blending the low- and high-molecular weight of PEGDA polymer improved the mechanical properties of the final hydrogel. All the hydrogel samples were non-cytotoxic up to 72 h after cell culturing. The system shows rapid blood hemostasis and promotes adhesive and cohesive strength matching targeted tissue properties with an applicating methodology compatible with surgical conditions. The developed SALS approach to optimize nanoparticles' microstructures in bio-adhesive applies to virtually any optically transparent nanocomposite and any type of anisotropic nanoparticles. As such, this method enables rational design of bio-adhesives with enhanced anisotropic mechanical properties which can be tailored to potentially any type of tissue.

Nanoparticle-based nanocomposite coatings with postprocessing for enhanced antimicrobial capacity of polymeric film.

Bacterial adhesion and biofilm formation on surfaces pose a significant risk of microbial contamination and chronic diseases, leading to potential health complications. To mitigate this concern, the implementation of antibacterial coatings becomes paramount in reducing pathogen propagation on contaminated surfaces. To address this requirement, our study focuses on developing cost-effective and sustainable methods using polymer composite coatings. Copper and titanium dioxide nanoparticles were used to assess their active antimicrobial functions. After coating the surface with nanoparticles, four different combinations of two postprocessing treatments were performed. Intense pulsed light was utilized to sinter the coatings further, and plasma etching was applied to manipulate the physical properties of the nanocomposite-coated sheet surface. Bacterial viability was comparatively analyzed at four different time points (0, 30, 60, and 120 min) upon contact with the nanocomposite coatings. The samples with nanoparticle coatings and postprocessing treatments showed an above-average 84.82% mortality rate at 30 min and an average of 89.77% mortality rate at 120 min of contact. In contrast, the control sample, without nanoparticle coatings and postprocessing treatments, showed a 95% microbe viability after 120 min of contact. Through this study, we gained critical insights into effective strategies for preventing the spread of microorganisms on high-touch surfaces, thereby contributing to the advancement of sustainable antimicrobial coatings.

Development of Antifreezing, Printable, Adhesive, Tough, Biocompatible, High-Water Content Hydrogel for Versatile Applications.

Hydrogels with different functionalities such as printability, antifreezing properties, adhesion, biocompatibility, and toughness are being continually developed. However, it has been extremely challenging to design adhesive, antifreezing, tough, and biocompatible multifunctional hydrogels with complex shapes simultaneously and prepare them in a short period. In this paper, novel composite hydrogels, which consist of poly(vinyl alcohol) grafted with styrylpyridinium group (PVA-SbQ) and TEMPO-oxidized cellulose nanofibrils (CNF), were successfully synthesized via UV photo-cross-linking. In addition to UV photo-cross-linking, the PVA-SbQ/CNF hydrogels with different shapes could be rapidly printed by facile visible light-based stereolithography printing and laser direct-writing without any photoinitiators in 3 min and 30 s, respectively. The results show that PVA-SbQ/CNF hydrogels are biocompatible because there are no photoinitiators and cross-linkers required during the printing process under visible light. Moreover, the adhesive, antifreezing, mechanical properties, and water-binding capacity of PVA-SbQ/CNF with high-water contents improved significantly as the CNF contents increased. Such hydrogels, which combine multiple advantages, present great potential for application in wound dressings and portable devices with specific requirements for shapes, adhesion, toughness, and tolerance in extreme environments such as dry environments and low temperatures.

Recent Advances in Organ-on-Chips Integrated with Bioprinting Technologies for Drug Screening.

Currently, the demand for more reliable drug screening devices has made scientists and researchers develop novel potential approaches to offer an alternative to animal studies. Organ-on-chips are newly emerged platforms for drug screening and disease metabolism investigation. These microfluidic devices attempt to recapitulate the physiological and biological properties of different organs and tissues using human-derived cells. Recently, the synergistic combination of additive manufacturing and microfluidics has shown a promising impact on improving a wide array of biological models. In this review, different methods are classified using bioprinting to achieve the relevant biomimetic models in organ-on-chips, boosting the efficiency of these devices to produce more reliable data for drug investigations. In addition to the tissue models, the influence of additive manufacturing on microfluidic chip fabrication is discussed, and their biomedical applications are reviewed.

Visible light laser direct-writing of high-resolution, biocompatible, super-multifunctional and tough hydrogels without photoinitiators in 30 s.

Currently, the lack of bioinks and long printing time limits the further development of biofabrication. Here we report a novel biocompatible, multi-functional and tough 3D printable hydrogel via visible light photocrosslinking of polyvinyl alcohol bearing styrylpyridinium group (PVA-SbQ). The high-resolution PVA-SbQ hydrogels with different designed shapes can be generated via laser direct-writing in 30 s without extra toxic crosslinkers or photoinitiators, and demonstrates excellent biocompatibility. The rapid laser direct-writing technology also results in a super-strong, tough hydrogel with excellent adhesive, swelling, self-healing, and photo-tunable properties due to the photodimerization of styrylpyridinium (SbQ) groups and the left-over massive amount of free hydroxyl groups in the hydrogel. For example, the maximum tensile strength, elongation, compressive strength adhesive strength of printed PVA-SbQ hydrogels can reach 1.0 MPa, 810 %, 33 MPa, 31 kPa, and 25,000 % respectively. And these properties can be adjusted by controlling the parameters for laser direct-writing. In addition, the introduced nitrogen cations by SbQ groups further endow hydrogels with the potential to develop novel functionality, which is demonstrated by integrating negatively charged nanocelluloses in the PVA-SbQ system to develop underwater adhesives, anti-freezing (-24.9 °C), and anti-bacterial hydrogels. This discovery opens multiple doors for developing PVA-SbQ based multi-functional hydrogel for various applications including biofabrication and tissue engineering.

Closed-loop vasculature network design for bioprinting large, solid tissue scaffolds.

Vascularization is an indispensable requirement for fabricating large solid tissues and organs. The natural vasculature derived from medical imaging modalities for large tissues and organs are highly complex and convoluted. However, the present bioprinting capabilities limit the fabrication of such complex natural vascular networks. Simplified bioprinted vascular networks, on the other hand, lack the capability to sustain large solid tissues. This work proposes a generalized and adaptable numerical model to design the vasculature by utilizing the tissue/organ anatomy. Starting with processing the patient's medical images, organ structure, tissue-specific cues, and key vasculature tethers are determined. An open-source abdomen magnetic resonance image dataset was used in this work. The extracted properties and cues are then used in a mathematical model for guiding the vascular network formation comprising arterial and venous networks. Next, the generated three-dimensional networks are used to simulate the nutrient transport and consumption within the organ over time and the regions deprived of the nutrients are identified. These regions provide cues to evolve and optimize the vasculature in an iterative manner to ensure the availability of the nutrient transport throughout the bioprinted scaffolds. The mass transport of six components of cell culture media-glucose, glycine, glutamine, riboflavin, human serum albumin, and oxygen was studied within the organ with designed vasculature. As the vascular structure underwent iterations, the organ regions deprived of these key components decreased significantly highlighting the increase in structural complexity and efficacy of the designed vasculature. The numerical method presented in this work offers a valuable tool for designing vascular scaffolds to guide the cell growth and maturation of the bioprinted tissues for faster regeneration post bioprinting.

The Effect of Crosslinking Degree of Hydrogels on Hydrogel Adhesion.

The development of adhesive hydrogel materials has brought numerous advances to biomedical engineering. Hydrogel adhesion has drawn much attention in research and applications. In this paper, the study of hydrogel adhesion is no longer limited to the surface of hydrogels. Here, the effect of the internal crosslinking degree of hydrogels prepared by different methods on hydrogel adhesion was explored to find the generality. The results show that with the increase in crosslinking degree, the hydrogel adhesion decreased significantly due to the limitation of segment mobility. Moreover, two simple strategies to improve hydrogel adhesion generated by hydrogen bonding were proposed. One was to keep the functional groups used for hydrogel adhesion and the other was to enhance the flexibility of polymer chains that make up hydrogels. We hope this study can provide another approach for improving the hydrogel adhesion generated by hydrogen bonding.

Tunable metacrylated hyaluronic acid-based hybrid bioinks for stereolithography 3D bioprinting.

Hyaluronic acid is a native extra-cellular matrix derivative that promises unique properties, such as anti-inflammatory response and cell-signaling with tissue-specific applications under its bioactive properties. Here, we investigate the importance of the duration of synthesis to obtain photocrosslinkable methacrylated hyaluronic acid (MeHA) with high degree of substitution. MeHA with high degree of substitution can result in rapid photocrosslinking and can be used as a bioink for stereolithographic (SLA) three dimensional 3D bioprinting. Increased degree of substitution results Our findings show that a ten-day synthesis results in an 88% degree of methacrylation (DM), whereas three-day and five-day syntheses result in 32% and 42% DM, respectively. The rheological characterization revealed an increased rate of photopolymerization with increasing DM. Further, we developed a hybrid bioink to overcome the non-cell-adhesive nature of MeHA by combining it with gelatin methacryloyl (GelMA) to fabricate 3D cell-laden hydrogel scaffolds. The hybrid bioink exhibited a 55% enhancement in stiffness compared to MeHA only and enabled cell-adhesion while maintaining high cell viability. Investigations also revealed that the hybrid bioink was a more suitable candidate for stereolithography (SLA) 3D bioprinting than MeHA because of its mechanical strength, printability, and cell-adhesive nature. This research lays out a firm foundation for the development of a stable hybrid bioink with MeHA and GelMA for first-ever use with SLA 3D bioprinting.

A rapid near-patient detection system for SARS-CoV-2 using saliva.

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

Designing Gelatin Methacryloyl (GelMA)-Based Bioinks for Visible Light Stereolithographic 3D Biofabrication.

Bioinks play a key role in determining the capability of the biofabricatoin processes and the resolution of the printed constructs. Excellent biocompatibility, tunable physical properties, and ease of chemical or biological modifications of gelatin methacryloyl (GelMA) have made it an attractive choice as bioinks for biomanufacturing of various tissues or organs. However, the current preparation methods for GelMA-based bioinks lack the ability to tailor their physical properties for desired bioprinting methods. Inherently, GelMA prepolymer solution exhibits a fast sol-gel transition at room temperature, which is a hurdle for its use in stereolithography (SLA) bioprinting. Here, synthesis parameters are optimized such as solvents, pH, and reaction time to develop GelMA bioinks which have a slow sol-gel transition at room temperature and visible light crosslinkable functions. A total of eight GelMA combinations are identified as suitable for digital light processing (DLP)-based SLA (DLP-SLA) bioprinting through systematic characterizations of their physical and rheological properties. Out of various types of GelMA, those synthesized in reverse osmosis (RO) purified water (referred to as RO-GelMA) are regarded as most suitable to achieve high DLP-SLA printing resolution. RO-GelMA-based bioinks are also found to be biocompatible showing high survival rates of encapsulated cells in the photocrosslinked gels. Additionally, the astrocytes and fibroblasts are observed to grow and integrate well within the bioprinted constructs. The bioink's superior physical and photocrosslinking properties offer pathways of tuning the scaffold microenvironment and highlight the applicability of developed GelMA bioinks in various tissue engineering and regenerative medicine applications.

Polyether ether ketone surface modification with plasma and gelatin for enhancing cell attachment.

Polyether ether ketone (PEEK) has shown great promise for implant and biomedical applications because of its excellent chemical, mechanical, and biocompatible properties. However, PEEK is bioinert, which causes weak cell adhesion and limits its use for biomedical applications such as bone implants. Therefore, the activation of the PEEK's surface for cell attachment is desirable. In this study, oxygen plasma and gelatin were used to modify PEEK's surface and the effects of surface roughness, wettability, and cell adhesion to the surface were studied. Surface roughness was measured using a laser scanning confocal microscope, and wettability was measured using the sessile drop method. There was no significant difference in the roughness of the three samples. The gelatin-coated surface showed higher wettability than the plasma-modified or control samples. The cell attachment and proliferation rate were assessed by scanning electron microscopy and the XTT assay, respectively. The XTT assay results indicated that a greater number of cells grew on the gelatin-coated PEEK surface than on the control or plasma-treated surfaces. These results confirmed that the plasma and gelatin treatments enhanced the biocompatibility of the PEEK samples. The increase in biocompatibility could make PEEK a better material candidate for treating bone related injuries and defects.

Increased sanitization potency of hydrogen peroxide with synergistic O3 and intense pulsed light for non-woven polypropylene.

Supplies of respiratory masks have recently become a concern due to the onset of the SARS-CoV-2 pandemic. Sanitization and reuse of masks can alleviate high mask consumption and production stresses. In the present work, improved sanitization potency of vaporous hydrogen peroxide (VHP) treatment of resilient bacterial spores while retaining polymeric filter performance was explored. A batch fumigation chamber with hydrogen peroxide (H2O2) vapor and ozone (O3) is featured, followed by intense pulsed light (IPL) flash treatments. A resilient bacterial indicator, Geobacillus stearothermophilus (G. stearothermophilus), was utilized to compare the efficacy of various H2O2 concentrations in combination with O3 and IPL. It was found that exposure to 30 minutes of 4.01 L min-1 0.03% H2O2 aqueous vapor and 3 g h-1 O3 followed by 10 IPL flashes per side completely inactivated G. stearothermophilus. The xenon sourced IPL irradiation was found to synergistically enhance radical production and strengthen the complementary biocidal interaction of H2O2 with O3. Due to the synergistic effects, H2O2 was able to sanitize at a diluted concentration of 0.03% H2O2. The physical properties, such as surface potential, tensile strength, hydrophobicity, and filtration efficiency of >300 nm saline water aerosol of fibrous polypropylene (PP) sheets, were maintained. In addition, no residue of sanitizers was detected, thus confirming the biosafety and applicability of this method to disposable masks. Performance was benchmarked and compared with commercially available processes. The synergistic regime was found to achieve sterilization of G. stearothermophilus at drastically reduced H2O2 concentrations and in ambient conditions relative to commercial methods.

Stereolithography 3D Bioprinting Method for Fabrication of Human Corneal Stroma Equivalent.

3D bioprinting technology is a promising approach for corneal stromal tissue regeneration. In this study, gelatin methacrylate (GelMA) mixed with corneal stromal cells was used as a bioink. The visible light-based stereolithography (SLA) 3D bioprinting method was utilized to print the anatomically similar dome-shaped structure of the human corneal stroma. Two different concentrations of GelMA macromer (7.5 and 12.5%) were tested for corneal stroma bioprinting. Due to high macromer concentrations, 12.5% GelMA was stiffer than 7.5% GelMA, which made it easier to handle. In terms of water content and optical transmittance of the bioprinted scaffolds, we observed that scaffold with 12.5% GelMA concentration was closer to the native corneal stroma tissue. Subsequently, cell proliferation, gene and protein expression of human corneal stromal cells encapsulated in the bioprinted scaffolds were investigated. Cytocompatibility in 12.5% GelMA scaffolds was observed to be 81.86 and 156.11% at day 1 and 7, respectively, which were significantly higher than those in 7.5% GelMA scaffolds. Elongated corneal stromal cells were observed in 12.5% GelMA samples after 7 days, indicating the cell attachment, growth, and integration within the scaffold. The gene expression of collagen type I, lumican and keratan sulfate increased over time for the cells cultured in 12.5% GelMA scaffolds as compared to those cultured on the plastic tissue culture plate. The expression of collagen type I and lumican were also visualized using immunohistochemistry after 28 days. These findings imply that the SLA 3D bioprinting method with GelMA hydrogel bioinks is a promising approach for corneal stroma tissue biofabrication.

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform.

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low-throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high-throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet-bioprinting technique for printing cell-laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano-responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.

Stereolithography 3D Bioprinting.

Stereolithography (SLA) 3D bioprinting has emerged as a prominent bioprinting method addressing the requirements of complex tissue fabrication. This chapter addresses the advancement in SLA 3D bioprinting in concurrent with the development of novel photocrosslinkable biomaterials with enhanced physical and chemical properties. We discuss the cytocompatible photoinitiators operating in the wide spectrum of the ultraviolet (UV) and the visible light and high-resolution dynamic mask projection systems with a suitable illumination source. The potential of SLA 3D bioprinting has been explored in various themes, like bone and neural tissue engineering and in the development of controlled microenvironments to study cell behavior. The flexible design and versatility of SLA bioprinting makes it an attractive bioprinting process with myriad possibilities and clinical applications.

An integrated microfluidic flow-focusing platform for on-chip fabrication and filtration of cell-laden microgels.

We present the development of a stable continuous, and integrated microfluidic platform for the high-throughput fabrication of monodisperse cell-laden microgel droplets with high and maintained cellular viability. This is through combining onto one chip all the required processes from the droplet generation in a flow focusing microfluidic junction passing through on-chip photocrosslinking to the separation of the droplets from the continuous oil phase. To avoid cellular aggregation during the droplet generation process, cells were treated with bovine serum albumin (BSA) before mixing with gelatin methacrylate (GelMA). And, a magnetic mixer was applied to the GelMA prepolymer-cell suspension syringe to eliminate cell sedimentation. These approaches resulted in having a reasonable distribution of cells among monodisperse microdroplets. The microdroplets were irradiated with a 405 nm wavelength laser beam while passing through the crosslinking chamber of the microfluidic device. The produced microgels enter the filtration unit of the same device where they were gently separated from the oil phase into the washing buffer aqueous solution of Tween 80 using the filter microposts array. The viability of the encapsulated cells was around 85% at day 1 and was maintained throughout 5 days. Using this method of controlling cell encapsulation with on-chip crosslinking and oil filtration, highly efficient cell-laden microgel production is achieved. The presented integrated microfluidic platform can be a candidate for standard cell-encapsulation experiments and other tissue engineering applications.

Visible Light Photoinitiation of Cell-Adhesive Gelatin Methacryloyl Hydrogels for Stereolithography 3D Bioprinting.

We present the first cell-attachable and visible-light-crosslinkable bioinks based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography three-dimensional (3D) bioprinting. To develop a visible-light-crosslinkable hydrogel, we systematically studied five combinations of GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentrations on the characteristics of the hydrogel. Experimental results show that the compressive Young's modulus and pore size are positively affected by the concentration of EY, whereas the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps in improving the compressive Young's modulus and cell attachment. We further employed the developed visible-light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.

An anatomical insight into the third head of biceps brachii muscle.

BACKGROUND: The biceps brachii muscle is known to show variations in the number of heads. This study was performed to evaluate the variations in the origin of the biceps brachii muscle heads. MATERIALS AND METHODS: Both extremities of 48 formalin fixed cadavers (n = 96) were studied for abnormal heads of biceps brachii muscle. The flexor compartment of the arm was dissected and the attachments of the biceps brachii muscle were studied in detail. Appropriate photographs were taken. RESULTS: Among ninety six upper limbs studied, we observed three heads of biceps on both sides of a male cadaver aged 56 yrs (3.33%). The two heads of the biceps arose from its usual position but the anomalous third head arose from the anterior limb of the 'V' shaped insertion of the deltoid muscle on the humerus. The third head was found to fuse with the common belly of the muscle well before the bicipital tendon and its aponeurosis. Bilaterally this third head was supplied by a twig of the musculocutaneous nerve. No other abnormalities relating to the biceps were observed in any of the sides of the other 47 cadavers (n = 94). CONCLUSION: Our study revealed that the incidence of third head of biceps brachii may be approximately 3.33 % but larger studies are needed to confirm this fact. The third head of biceps brachii may be an incidental finding at autopsy or during routine anatomical dissections. Unless symptomatic, the third head of biceps brachii may not be detected in clinical studies (Fig. 3, Ref. 15).

Anomalous pectoral musculature.

Anomalous disposition of pectoral muscles was encountered in an adult female cadaver on the left side. A prominent cleft separating the sternocostal and clavicular portions of the pectoralis major was noticed. The fibers of pectoralis major were partially fused with the deltoid, resulting in obliteration of the deltopectoral groove. Interestingly, cephalic vein was seen traversing superficial to the clavicular portion of the pectoralis major and pierced it to drain into the axillary vein. The pectoralis minor was inserted mainly on the coracoid process and few fibers were found blending with the coracobrachialis and short head of biceps brachii. Further, pectoralis minimus, a rare anatomic variant, was also observed lying superior to pectoralis minor. It was innervated by a twig from the lateral pectoral nerve at its superficial surface. Awareness of possibility of such anomalous muscles is important for surgeons operating on the chest wall.